Transcription factors that regulate nicotine biosynthesis in tobacco

ABSTRACT

The present invention provides nucleic acids encoding transcription factors and methods of using these nucleic acids to modulate nicotine production in plants and to produce plants having modulated nicotine production.

RELATED APPLICATION INFORMATION

This application is a divisional application of U.S. patent applicationSer. No. 16/059,696, filed Aug. 9, 2018, which claims priority to U.S.patent application Ser. No. 15/412,443, filed Jan. 23, 2017, whichclaims priority to U.S. Pat. No. 9,580,722, issued on Feb. 28, 2017,which claims the benefit of U.S. Provisional Application Ser. No.61/771,526, filed Mar. 1, 2013, the disclosures of each of which areincorporated by reference herein in their entireties.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 5051-826TS_ST25.txt, 37,572 bytes in size, generated onFeb. 20, 2014 and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

FIELD OF THE INVENTION

The present invention relates to transcription factors and their use inmodulation of nicotine biosynthesis in plants.

BACKGROUND OF THE INVENTION

Nicotine is found predominantly in the genus Nicotiana and in lowquantities in some other species of the family Solanaceae (Sheen S J(1988) J Food Science 53: 1572-1573). Nicotine is the most abundantalkaloid in commercial tobacco (N. tabacum L.) cultivars (Siminszky etal. (2005) Proc Natl Acad Sci USA 102: 14919-14924), and naturally playsan important role in tobacco resistance to insect herbivores (Steppuhnet al (2004) PLoS Biology 2: 1074-1080). Nicotine is synthesized intobacco root tip and transported to leaf and stored in leaf cell vacuoleby a multidrug and toxic compound extrusion (MATE) transporter (Moritaet al. (2009) Proc Natl Acad Sci 106: 2447-2452). The pathway fornicotine biosynthesis involves convergence of two biosyntheticallydistinct branches. The enzymes and the genes encoding these enzymesinvolved in the pathway have been identified except for the enzyme/genethat catalyzes the final condensation step of nicotinic acid andmethyl-pyrrolinium cation to form nicotine, generally referred to asnicotine synthase. It has been proposed that a reduction reaction occursfollowed by oxidation of nicotinic acid prior to formation of specificpyridine moiety that condenses with the methylpyrrolinium cation to formnicotine (Friesen and Leete (1990) Tetrahedron Letters 31:6295-6298).A622 and NBB1 have been proposed as catalysts of the final steps ofnicotine biosynthesis (Hibi et al. (1994) Plant Cell 6:723-35; Shoji etal. (2002) Plant Mol Biol 50:427-440; Hashimoto and Kato 2007; Kajikawaet al. (2009) Plant Mol Biol 69: 287-298; Kajikawa et al. (2011) PlantPhysiol 155(4):2010-22). Putrescine N-methyltransferase (PMT) andquinolinic acid phosphoribosyltransferase (QPT), the first committingenzymes in each branch of the nicotine biosynthetic pathway, arebelieved to be the key enzymes in nicotine production (Feth et al.(1986) Planta 168: 402-407; Wagner et al. (1986) Physiol Plantarum 68:667-672). A few minor alkaloids, including nornicotine, anabasine, andanatabine, are also synthesized in this pathway, with nornicotine beingdirectly converted from nicotine by nicotine demethylase whosecorresponding genes have been cloned (Siminszky et al. (2005) Proc NatlAcad Sci USA 102: 14919-14924).

This invention addresses the need for compositions and methods thatmodulate the nicotine biosynthesis pathway in plants.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence is: (a) the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7; (b) anucleotide sequence that is at least 95% identical to the nucleotidesequence of (a) above; (c) a nucleotide sequence that encodes atranscription factor that modulates nicotine biosynthesis having theamino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ IDNO:8; (d) a nucleotide sequence that differs from the nucleotidesequence of (a), (b) or (c) above due to the degeneracy of the geneticcode; (e) a nucleotide sequence which anneals under stringenthybridization conditions to the nucleotide sequence of (a), (b), (c) or(d) above, or a complement thereof; or (f) any combination of thenucleotide sequences of (a)-(e) above.

In a second aspect, a nucleic acid construct is provided, the nucleicacid construct comprising in the 5′ to 3′ direction, a promoter operablein a plant cell and a nucleic acid molecule of the invention positioneddownstream from said promoter and operatively associated therewith. Insome aspects, the nucleic acid molecule is positioned in the sensedirection and in other aspects the nucleic acid molecule is positionedin the antisense direction.

In a third aspect, a nucleic acid construct comprising double strandedRNA molecule comprising an antisense strand and a sense strand isprovided, wherein the nucleotide sequence of the antisense strand iscomplementary to a portion of a nucleic acid molecule of the invention.

In an additional aspect, a method of identifying a plant having amutation in a gene encoding a transcription factor that modulatesnicotine biosynthesis is provided, comprising screening a population ofplants by high-throughput DNA sequence analysis using primers comprisingone or more portions of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and/or SEQID NO:7, and/or by TILLING (Targeting Induced Local Lesions In Genomes)using a probe comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and/orSEQ ID NO:7 or a portion of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and/orSEQ ID NO:7, thereby identifying a plant having a mutation in a geneencoding a transcription factor that modulates nicotine biosynthesis.

A further aspect of the invention provides a method of producing a planthaving a mutation in a gene encoding a transcription factor thatmodulates nicotine biosynthesis, comprising targeted mutagenesis using areagent comprising a nucleic acid consisting of a portion of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5 and/or SEQ ID NO:7.

A further aspect of the invention provides a nucleic acid constructcomprising in the 5′ to 3′ direction, a plant promoter and a nucleicacid molecule of the invention or a fragment thereof positioneddownstream from said promoter and operatively associated therewith, saidnucleic acid molecule in antisense orientation.

In an additional aspect, a method of identifying a plant having amutation in a gene encoding a transcription factor that modulates (e.g.,regulates) nicotine biosynthesis is provided, comprising screening apopulation of plants by high-throughput DNA sequence analysis usingprimers comprising one or more portions of SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5 and/or SEQ ID NO:7, and/or by TILLING (Targeting Induced LocalLesions In Genomes) using a probe comprising SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5 and/or SEQ ID NO:7 or a portion of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5 and/or SEQ ID NO:7, thereby identifying a plant having amutation in a gene encoding a transcription factor that modulates (e.g.,regulates) nicotine biosynthesis.

A further aspect of the invention provides a method of producing a planthaving a mutation in a gene encoding a transcription factor thatmodulates nicotine biosynthesis, comprising targeted mutagenesis using areagent comprising a nucleic acid consisting of a portion of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5 and/or SEQ ID NO:7, thereby producing aplant having a mutation in a gene encoding a transcription factor thatmodulates nicotine biosynthesis.

The present invention further provides vectors and expression cassettescomprising at least one nucleic acid construct of the invention.

In still other aspects, the present invention provides a method ofproducing a plant having modulated nicotine content, comprisingintroducing into a plant cell a nucleic acid construct of the inventionto produce a transgenic plant cell comprising said nucleic acidconstruct; and regenerating said transgenic plant cell to produce atransgenic plant comprising said nucleic acid construct. In someaspects, the plant is a tobacco plant.

In an additional aspect, a method of modulating nicotine content in aplant is provided, the method comprising introducing into a plant cell anucleic acid construct of the present invention to produce a transgenicplant cell comprising said nucleic acid construct; and regenerating saidtransgenic plant cell to produce a transgenic plant comprising saidnucleic acid construct, thereby modulating nicotine production in saidtransgenic plant. In aspects, the plant is a tobacco plant.

The present invention further provides bacterial cells, plants and plantparts thereof comprising at least one nucleic acid construct of theinvention as well as well as crops and products produced from saidplants and parts thereof.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a vector map of T-DNA region of pBI121.

FIG. 2 provides a schematic representation of the region of vector pQLiused for inverted repeat cloning. Gus linker: partial sequence from GUSgene.

FIG. 3 shows organ expression pattern of NtMYC2.

FIG. 4 shows NtMYC2 expression in tobacco root after topping, wounding,or MeJA treatment. Northern hybridization shows a time course inductionpattern of NtMYC2 after each treatment. The probe is from NtMYC2b. The25S rRNA stained in gel with EtBr is shown as a loading reference.

FIG. 5 shows binding of NtMYC2a to the G2-box in NtQPT2 promoter. Lane1, no protein; lane 2, NtMYC2aΔN+probe; lane 3, NtMYC2aΔN+probe+500×coldprobe; lane 4, NtMYC2aΔN+probe+500×mutated cold probe. Arrows indicatethe shifted (migrate slower) and free probes, respectively.

FIG. 6 shows expression of NtMYC2a and NtMYC2b in NtMYC2a or NtMYC2bover-expression lines. Northern hybridization shows NtMYC2 mRNA level inNtMYC2a or NtMYC2b over-expression lines. The bottom panel shows theribosomal RNA in gel stained with EtBr as an RNA loading reference. Thewild-type and vector control are also shown.

FIG. 7 shows the combined expression of NtMYC2a and NtMYC2b in NtMYC2RNAi lines. Top panel: Northern hybridization of NtMYC2 mRNA level inNtMYC2 RNAi lines. The bottom panel shows ribosomal RNA in gel stainedwith EtBr as an RNA loading reference. The wild-type and vector controlare also shown.

FIG. 8 shows nicotine concentration of over-expression and RNAi lines ofNtMYC2a and NtMYC2b. DW: dry weight.

FIG. 9 shows QPT expression level in NtMYC2a, or b over-expression andRNAi transgenic tobacco lines. Northern blot hybridization uses partialQPT coding sequence as probes. The rRNA stained with EtBr in gel isshown at the lower panel as an RNA loading reference.

FIG. 10 shows PMT expression in NtMYC2a, or b over-expression and RNAitransgenic lines. Northern blot hybridization using partial PMT codingsequence as probes. The rRNA stained with EtBr in gel is shown in thelower panel as an RNA loading reference.

FIGS. 11A-11B show expression of nicotine biosynthesis pathway genesmeasured by qRT-PCR. The expression level in the vector control was setto 1, and expression of pathway genes in T₀ transgenic plants (FIG. 11A)and T₁ plants (FIG. 11B) is shown relative to this level. The averageexpression level of two transformation events from each construct thataffects the nicotine level most is shown; the error bars indicatestandard errors.

FIG. 12 shows leaf nicotine levels in T₁ transgenic plants undernon-topping and topping treatments. DW %: dry weight percentage. Valuesare average of T₁ transgenic plants (PCR positive, n=15-30) from eachgroup with standard errors. Different letters indicate significantdifference within each category (t-test, p<0.05).

FIG. 13 shows nornicotine, anabasine and anatabine levels in leaf of T1transgenic plants. DW %: dry weight percentage. Values are average of T1transgenic plants (PCR positive, n=15-30) from each group with standarddeviations. Different letters indicate significant difference withineach category (t-test, p<0.05).

FIG. 14 NtERF98 expression in four organs of tobacco plant. Northernblot hybridization of total RNA isolated from various organs of fullygrown plant was probed with a partial NtERF98 coding sequence. The 25SrRNA stained with EtBr in gel was also shown as a loading reference.

FIG. 15 shows a time course of NtERF98 expression in tobacco roots aftertopping, wounding, or MeJA treatment. Northern blot was hybridized withNtERF98 coding sequence as a probe, and 25S rRNA with EtBr staining as aloading reference.

FIG. 16 shows NtERF98 and QPT expression in ten putative NtERF98over-expression transgenic plants. Northern hybridization of QPT (top)and NtERF98 mRNA (middle) is shown. 25S rRNA stained with EtBr is alsoshown as a loading reference (bottom).

FIG. 17 shows nicotine concentration in ten NtERF98 over-expressiontransformed tobacco lines and wild type control. DW: dry weight.

FIG. 18 shows Northern analysis of NtERF98 expression in its RNAitransgenic lines. The 25S rRNA stained with EtBr is shown as a loadingreference.

FIG. 19 shows Northern hybridization of QPT expression in the NtERF98RNAi transformed tobacco lines. The 25S rRNA stained with EtBr is shownin the lower panel as a loading reference.

FIG. 20 shows nicotine concentration in NtERF98 RNAi transgenic tobaccolines and wild type control. DW: dry weight.

FIG. 21 shows a Northern blot analysis of NtETTa expressions in tobaccoroot, stem, leaf, and flower. The 25S rRNA stained with EtBr on the gelis shown as a loading reference.

FIG. 22 shows a time course of NtETTa expression response in tobaccoroot after topping, wounding, or MeJA treatment. Shown are the Northernblot hybridization of NtETTa (top panel) and the 25S rRNA stained withEtBr as a loading reference (bottom panel).

FIG. 23 shows expression levels of NtETTa in over-expression lines andRNAi lines. Shown are Northern hybridization of NtETTa (top panel) andthe 25S rRNA stained with EtBr as a loading reference (bottom panel).

FIG. 24 shows QPT expressions in NtETTa over-expression and RNAi lines.Shown are Northern blot hybridization of QPT (top panel) and the 25SrRNA stained with EtBr as a loading reference (bottom panel).

FIG. 25 shows nicotine concentrations of NtETTa over-expression and RNAilines. The line numbers are the same as in the previous figures. DW: dryweight.

FIGS. 26A-26C show total alkaloid levels (% dry weight (DW)) ingreenhouse grown T3 plants. Leaf samples were taken 13 weeks aftertransplanting and prior to topping, and at 17 and 31 days after topping.Two sets of control plants were included, a line transformed with a(VC=vector containing no MYC2 coding sequence), and VC×NT=plants fromseed from a cross of the vector control with untransformed tobacco.

FIGS. 27A-27C show total alkaloid levels (% dry weight (DW)) in fieldgrown T3 plants. Leaf samples were taken 13 weeks after transplantingand prior to topping, and at 17 and 31 days after topping. Two sets ofcontrol plants were included, a line transformed with a (VC=vectorcontaining no MYC2 coding sequence), and VC×NT=plants from seed from across of the vector control with untransformed tobacco.

DETAILED DESCRIPTION OF THE INVENTION

This description is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. Thus, theinvention contemplates that in some embodiments of the invention, anyfeature or combination of features set forth herein can be excluded oromitted. In addition, numerous variations and additions to the variousembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention. Hence, the following descriptions are intended toillustrate some particular embodiments of the invention, and not toexhaustively specify all permutations, combinations and variationsthereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents and other referencescited herein are incorporated by reference in their entireties for theteachings relevant to the sentence and/or paragraph in which thereference is presented. References to techniques employed herein areintended to refer to the techniques as commonly understood in the art,including variations on those techniques or substitutions of equivalenttechniques that would be apparent to one of skill in the art.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a composition comprises components A, Band C, it is specifically intended that any of A, B or C, or acombination thereof, can be omitted and disclaimed singularly or in anycombination.

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The term “about,” as used herein when referring to a measurable valuesuch as a dosage or time period and the like, refers to variations of±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

As used herein, phrases such as “between X and Y” and “between about Xand Y” should be interpreted to include X and Y. As used herein, phrasessuch as “between about X and Y” mean “between about X and about Y” andphrases such as “from about X to Y” mean “from about X to about Y.”

The terms “comprise,” “comprises” and “comprising” as used herein,specify the presence of the stated features, integers, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, integers, steps,operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention. Thus, the term “consisting essentially of” when used in aclaim of this invention is not intended to be interpreted to beequivalent to “comprising.”

Nicotine biosynthesis occurs predominantly in the roots of tobaccoplants (Dawson RF (1941) Science 94 (2443):396-397; Dawson R F (1942)American Journal of Botany 29(10):813-815). The cortex and epidermis ofthe differentiated region of the root tip is considered the site ofnicotine production. The plant then transports the alkaloids through thevascular bundle to the leaves where the alkaloids are then stored in thevacuoles (Shoji et al. (2000) Plant & Cell Physiol 41: 831-839, Shoji etal. (2002) Plant Mol Biol 50: 427-440; Katoh et al. (2005) PlantBiotechnology 22: 389-392.). Several transporters may be involved in thetranslocation process. A transporter gene, named MATE, has recently beencloned and characterized (Morita et al. (2009) Proc Natl Acad Sci (USA)106: 2447-2452).

Biotic and abiotic stresses, such as herbivore damage, topping(decapitation of the apical meristem at an early stage of flowering) andsuckering (removing the axillary buds of plants activated by topping)can significantly increase nicotine accumulation in tobacco leaf(Baldwin I T (1988) Oecologia 77: 378-381; Wang et al. (2008) Nico JIntegr Plant Biol 50: 958-964). Nicotine biosynthesis and accumulationis mediated by endogenous phytohormone changes, which affect expressionof the genes involved in nicotine biosynthesis. So far, jasmonic acid(JA), ethylene, auxin and abscisic acid (ABA) have been shown to affectnicotine biosynthesis (Shoji et al. (2000) Plant Cell Physiol 41:1072-1076; Shi et al. (2006) J Exp Bot 57: 2899-2907; Lackman et al.(2011) Proc Natl Acad Sci USA 108: 5891-5896).

Nicotine production stimulated by JA treatment is well documented. Majornicotine biosynthetic pathway genes, including putrescineN-methyltransferase gene (NtPMT), quinolinic acidphosphoribosyltransferase gene (NtQPT), ornithine decarboxylase gene(NtODC), Arginine decarboxylase gene (NtADC), N-methylputrescine oxidasegene (NtMPO), an isoflavone reductase-like gene (NtA622) and a berberinebridge enzyme-like gene (NtBBL) have been shown to be up-regulated tovarious extents by MeJA application (Cane et al. (2005) Functional PlantBiology 32: 305; Katoh et al. (2007) Plant Cell Physiol 48: 550-554;U.S. Patent Application Publication 20070240728). Stimulation ofnicotine production by MeJA or wounding in tobacco is mediated by the JAsignaling pathway similar to that in Arabidopsis. Both NtCOI1(Coronatine Insensitive 1) expression and NtJAZ (Jasmonate Zim) proteindegradation are required for nicotine biosynthesis (Shoji et al. (2008)Plant Cell Physiol 49: 1003-1012).

Major steps in nicotine biosynthetic pathway have been elucidated in thepast two decades or so. However, studies on the regulation of thispathway at the transcription level have only begun recently. A tobaccotranscription factor database has been established based on in silicoanalysis of genomic sequencing data. More than 2500 transcriptionfactors from 64 families were identified (Rushton et al. (2008) PlantPhysiol 147: 280-295). However, which transcription factors are involvedin nicotine biosynthesis and how those transcription factors functiontogether to regulate nicotine biosynthetic pathway has remained unclear.

Genetic controls influence the type and level of alkaloids observed inNicotiana tabacum. In one system, two unlinked genetic loci, Nic1 andNic2 (or A and B), have been shown to control the total alkaloidaccumulation. Nic1 and Nic2 have different dosage effects on alkaloidaccumulation. Nic1 (A) has 2.4-fold greater activity than Nic2 (B) andtheir effects are additive (Legg and Collins (1971) Can. J. Genet. Cytol13: 287-291). Commercial varieties with high total alkaloid content areconsidered as homozygous dominant (AABB) at these two loci and lowalkaloid level genotypes are considered as homozygous recessive (aabb)at these two loci. Under this genetic model, nine genotypes of tobaccoplants with different levels of total alkaloids were developed (Legg etal. (1969) J Hered 60: 213-217; Legg and Collins (1971) Can. J. Genet.Cytol 13: 287-291). Very recently, the Nic2 locus has been molecularlycharacterized. It is actually a cluster of transcription factor genesfrom the ethylene responsive factor (ERF) family. They regulate nicotinebiosynthetic pathway genes (Shoji et al. (2010) The Plant Cell 22:3390-3409).

Another system controls the types of alkaloids produced and has agenetic locus which controls the conversion of nicotine to nornicotine.When both alleles of this locus are recessive, the plant containspredominantly nicotine. If one or both alleles are dominant, the plantprimarily produces nornicotine. Nornicotine results from demethylationof nicotine (Mann et al. (1964) Crop Sci. 4:349-353). The gene encodingnicotine demethylase, a cytochrome P450 gene, CYP82E4, was cloned andcharacterized (Siminszky et al. (2005) Proc Natl Acad Sci (USA) 102:14919-14924)). Suppressing the expression of this gene resulted in adrastic reduction in nornicotine content (Lewis et al. (2008) PlantBiotechnology Journal 6: 346-354).

In addition to the two genetic systems described above, some minor orquantitative factors are involved in alkaloid synthesis. Therefore, itis possible to produce tobacco lines with varying alkaloid contentwithin the range of the parents' alkaloid levels (Matzinger et al.(1972) Crop Sci. 12: 40-43, Matzinger et al. (1989) Crop Sci. 29:74-77).

The low nicotine content trait has been of interest to tobacco breeders.LA Burley 21 is a low total alkaloid line produced by incorporation of alow alkaloid gene(s) from a Cuban cigar variety into Burley 21 throughseveral backcrosses (Legg et al. (1970) Crop Sci 10: 212. It hasapproximately 0.2% total alkaloids (dry weight) compared to the 3.5%(dry weight) of its parent, Burley 21. Similarly, Chaplin and Burk(Agronomy Journal 75: 133-136 (1983)) developed some flue-cured tobaccolines with different alkaloid levels by backcrossing. They used NC95,SC58, and Coker 139 as recurrent parent lines and crossed them with LAFC53 (a low total alkaloid line in NC95 background) followed by severalbackcrosses. Five different alkaloid levels were obtained from the NC95family, six from the SC58 family, and four from the Coker139 family.

However, the breeding of commercial tobacco cultivars is morecomplicated than simply focusing on the alkaloid content in leaf. It hasbeen found that alkaloid accumulation is genetically linked with otherimportant agronomic traits. For example, a reverse correlation existsbetween total alkaloids and yield. Selection for increased yield mayresult in reduced level of total alkaloids (Chaplin and Week (1976) CropSci 16: 416-418). Conventional breeding methods have not been veryefficient at breaking this close genetic relationship. It is believedthat genetic manipulation at the molecular level may meet this need oftobacco breeders by only modifying alkaloid accumulation withoutaffecting other traits such as yield.

The present invention provides compositions and methods for the geneticmanipulation of alkaloid accumulation, more particularly nicotineaccumulation, in plants. Thus, in one aspect the present inventionprovides an isolated nucleic acid molecule comprising a nucleotidesequence is: (a) the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5 B SEQ ID NO:7; (b) a nucleotide sequence that is at least95% identical to the nucleotide sequence of (a) above; (c) a nucleotidesequence that encodes a transcription factor that modulates nicotinebiosynthesis having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6 or SEQ ID NO:8; (d) a nucleotide sequence that differs fromthe nucleotide sequence of (a), (b) or (c) above due to the degeneracyof the genetic code and that encodes a transcription factor thatmodulates nicotine biosynthesis; (e) a nucleotide sequence which annealsunder stringent hybridization conditions to the nucleotide sequence of(a), (b), (c) or (d) above, or a complement thereof; or (f) anycombination of the nucleotide sequences of (a)-(e) above. The nucleicacid molecules of the invention (the nucleotide sequences of SEQ ID NO:1(NtMYC2a cDNA), SEQ ID NO:3 (NtMYC2b cDNA), SEQ ID NO:5 (NtERF98 cDNA),and SEQ ID NO:7 (NtETTa cDNA)) encode transcription factors thatmodulate nicotine biosynthesis, their respective amino acid sequencesbeing SEQ ID NO:2 (NtMYC2a), SEQ ID NO:4 (NtMYC2b), SEQ ID NO:6(NtERF98) and SEQ ID NO:8 (NtETTa).

Thus, overexpression or reduced expression of an isolated nucleic acidmolecule or a nucleic acid construct described herein can result in theplant having increased or decreased nicotine content (as compared to awild type plant or a plant that does not comprise said isolated nucleicacid molecule or said nucleic acid construct). Thus, in one aspect, theinvention provides a recombinant nucleic acid molecule comprising one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and thelike) nucleotide sequences, each of which when expressed in a plantconfer increased or decreased nicotine content on said plant, whereinthe one or more nucleotide sequences comprise, consist essentially of,or consist of: (a) the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5 and/or SEQ ID NO:7; (b) a nucleotide sequence that is atleast 95% identical to the nucleotide sequence of (a) above; (c) anucleotide sequence that encodes a transcription factor that modulatesnicotine biosynthesis having the amino acid sequence of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6 and/or SEQ ID NO:8; (d) a nucleotide sequence thatdiffers from the nucleotide sequence of (a), (b) or (c) above due to thedegeneracy of the genetic code and that encodes a transcription factorthat modulates nicotine biosynthesis; (e) a nucleotide sequence whichanneals under stringent hybridization conditions to the nucleotidesequence of (a), (b), (c) or (d) above, or a complement thereof; or (f)any combination of the nucleotide sequences of (a)-(e) above.

The nucleic acid molecules/transcription factors of the presentinvention can be comprised in any construct useful for modulating oraltering nicotine content in an organism expressing said transcriptionfactors. Thus, for example, transcription factors of the invention canbe comprised in antisense constructs, RNAi constructs and the like.

Accordingly, in one embodiment, the present invention provides nucleicacid construct comprising in the 5′ to 3′ direction, a promoter operablein a plant cell and the nucleic acid molecule of the inventionpositioned downstream from said promoter and operatively associatedtherewith. In some embodiments, the nucleic acid molecule is positionedin the sense direction. In other embodiments, the nucleic acid moleculeis positioned in the antisense direction. In some embodiments, thepromoter can be a heterologous promoter.

In a still further embodiment, the present invention provides an RNAiconstruct comprising, consisting essentially of, or consisting of anucleic acid molecule of the invention. Thus, in some embodiments, anucleic acid construct comprising double stranded RNA moleculecomprising an antisense strand and a sense strand is provided, whereinthe nucleotide sequence of the antisense strand is complementary to aportion of the nucleic acid molecule of the invention. In someembodiments, the portion of the nucleic acid molecule of the inventioncomprises, consists essentially or consists of about 18 to about 1000consecutive nucleotides of said nucleic acid molecule, wherein thedouble stranded RNA molecule inhibits expression of a transcriptionfactor of the invention (e.g., nucleotide sequence of SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5 and/or SEQ ID NO:7) Thus, in some embodiments, theportion of the nucleic acid molecule of the invention to which theantisense strand is complementary comprises, consists essentially orconsists of about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610,620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750,760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890,900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 nucleotides andthe like or any range therein. Thus, in some embodiments, the portioncan be about 18 to about 25 consecutive nucleotides of said nucleic acidmolecule, or any range therein. In other embodiments, the portion can beabout 75 to about 1000 consecutive nucleotides of said nucleic acidmolecule, or any range therein. In still other embodiments, the portioncan be about 95 to about 900 consecutive nucleotides of said nucleicacid molecule, or any range therein. In an additional embodiment, theportion can be about 100 to about 500 consecutive nucleotides of saidnucleic acid molecule, or any range therein. Thus, in some embodiments,the dsRNA can comprise two identical (self-complementary) fragments ofvarying length (e.g., about 75 to about 900 contiguous nucleotides, orany range therein) of the target gene with a spacer in between (see,e.g., FIG. 2), which then forms a hairpin and can be cleaved intosmaller pieces that can then act to inhibit expression of the targetgene. In some embodiments, the spacer can be an intron (see. e.g.,Wesley et al. Plant J27(6):581-590 (2002).

Thus, in some embodiments, the RNAi construct produces a siRNA or miRNA.In other embodiments, the RNAi construct can be a short hairpin RNA(shRNA). In still other embodiments, the RNAi construct can be ahairpinRNA (hpRNA) construct. Methods for making such constructs arewell known in the art (see. e.g., Wesley et al. Plant J 27(6):581-590(2002)).

In a further embodiment, the present invention provides a nucleic acidconstruct comprising in the 5′ to 3′ direction, a plant promoter and anucleic acid molecule of the invention or a fragment thereof positioneddownstream from said promoter and operatively associated therewith, saidnucleic acid molecule in antisense orientation. A fragment of a nucleicacid molecule of the invention can be about 10 consecutive nucleotidesto the full size of the nucleic acid molecule (e.g., about 2500 bp).Thus, a fragment of a nucleic acid molecule of the invention can beabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860,870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, 2500 nucleotides and the like or any range therein.

In some embodiments of the invention, nucleotide sequences havingsignificant sequence identity to the nucleotide sequences of theinvention are provided. “Significant sequence identity” or “significantsequence similarity” means at least about 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, and/or 100% identity or similarity with another nucleotidesequence. Thus, in additional embodiments, “significant sequenceidentity” or “significant sequence similarity” means a range of about70% to about 100%, about 75% to about 100%, about 80% to about 100%,about 81% to about 100%, about 82% to about 100%, about 83% to about100%, about 84% to about 100%, about 85% to about 100%, about 86% toabout 100%, about 87% to about 100%, about 88% to about 100%, about 89%to about 100%, about 90% to about 100%, about 91% to about 100%, about92% to about 100%, about 93% to about 100%, about 94% to about 100%,about 95% to about 100%, about 96% to about 100%, about 97% to about100%, about 98% to about 100%, and/or about 99% to about 100% identityor similarity with another nucleotide sequence. Therefore, in someembodiments, a nucleotide sequence of the invention is a nucleotidesequence that has significant sequence identity to the nucleotidesequence of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and/or SEQ IDNO:7.

In some embodiments, a polypeptide of the invention comprises, consistsessentially of, or consists of an amino acid sequence that is at least70% identical, e.g., at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,and/or 100% identical to an amino acid sequence of any of SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:6, and/or SEQ ID NO:8.

In some embodiments, a polypeptide or nucleotide sequence of theinvention can be a conservatively modified variant. As used herein,“conservatively modified variant” refer to polypeptide and nucleotidesequences containing individual substitutions, deletions or additionsthat alter, add or delete a single amino acid or nucleotide or a smallpercentage of amino acids or nucleotides in the sequence, where thealteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.

As used herein, a conservatively modified variant of a polypeptide isbiologically active and therefore possesses the desired activity of thereference polypeptide (e.g., transcription factor activity; reducing orincreasing the nicotine content in a plant) as described herein. Thevariant can result from, for example, a genetic polymorphism or humanmanipulation. A biologically active variant of the reference polypeptidecan have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity or similarity (e.g., about 40% to about 99% or more sequenceidentity or similarity and any range therein) to the amino acid sequencefor the reference polypeptide as determined by sequence alignmentprograms and parameters described elsewhere herein. An active variantcan differ from the reference polypeptide sequence by as few as 1-15amino acid residues, as few as 1-10, such as 6-10, as few as 5, as fewas 4, 3, 2, or even 1 amino acid residue.

Naturally occurring variants may exist within a population. Suchvariants can be identified by using well-known molecular biologytechniques, such as the polymerase chain reaction (PCR), andhybridization as described below. Synthetically derived nucleotidesequences, for example, sequences generated by site-directed mutagenesisor PCR-mediated mutagenesis which still encode a polypeptide of theinvention, are also included as variants. One or more nucleotide oramino acid substitutions, additions, or deletions can be introduced intoa nucleotide or amino acid sequence disclosed herein, such that thesubstitutions, additions, or deletions are introduced into the encodedprotein. The additions (insertions) or deletions (truncations) may bemade at the N-terminal or C-terminal end of the native protein, or atone or more sites in the native protein. Similarly, a substitution ofone or more nucleotides or amino acids may be made at one or more sitesin the native protein.

For example, conservative amino acid substitutions may be made at one ormore predicted, preferably nonessential amino acid residues. A“nonessential” amino acid residue is a residue that can be altered fromthe wild-type sequence of a protein without altering the biologicalactivity, whereas an “essential” amino acid is required for biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue with a similarside chain. Families of amino acid residues having similar side chainsare known in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Suchsubstitutions would not be made for conserved amino acid residues, orfor amino acid residues residing within a conserved motif, where suchresidues are essential for protein activity.

For example, amino acid sequence variants of the reference polypeptidecan be prepared by mutating the nucleotide sequence encoding the enzyme.The resulting mutants can be expressed recombinantly in plants, andscreened for those that retain biological activity by assaying forincreased or reduced nicotine content using standard assay techniques asdescribed herein. Methods for mutagenesis and nucleotide sequencealterations are known in the art. See, e.g., Kunkel (1985) Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol.154:367-382; and Techniques in Molecular Biology (Walker & Gaastra eds.,MacMillan Publishing Co. 1983) and the references cited therein; as wellas U.S. Pat. No. 4,873,192. Clearly, the mutations made in the DNAencoding the variant must not disrupt the reading frame and preferablywill not create complimentary regions that could produce secondary mRNAstructure. See, EP Patent Application Publication No. 75,444. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1978) Atlas of Protein Sequence and Structure (NationalBiomedical Research Foundation, Washington, D.C.), herein incorporatedby reference.

The deletions, insertions and substitutions in the polypeptidesdescribed herein are not expected to produce radical changes in thecharacteristics of the polypeptide (e.g., the activity of thepolypeptide). However, when it is difficult to predict the exact effectof the substitution, deletion or insertion in advance of doing so, oneof skill in the art will appreciate that the effect can be evaluated byroutine screening assays that can screen for the particular polypeptideactivities of interest (e.g., conferring increased or reduced nicotinecontent to a plant).

In some embodiments, the compositions of the invention can compriseactive fragments of the polypeptide. As used herein, “fragment” means aportion of the reference polypeptide that retains the polypeptideactivity of conferring increased or decreased nicotine content in aplant. A fragment also means a portion of a nucleic acid moleculeencoding the reference polypeptide. An active fragment of thepolypeptide can be prepared, for example, by isolating a portion of apolypeptide-encoding nucleic acid molecule that is expressed to producethe encoded fragment of the polypeptide (e.g., by recombinant expressionin vitro), and assessing the activity of the fragment. Nucleic acidmolecules encoding such fragments can be at least about 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2000, 2100,2200, 2400 or 2500, contiguous nucleotides, or up to the number ofnucleotides present in a full-length polypeptide-encoding nucleic acidmolecule. As such, polypeptide fragments can be at least about 50, 60,70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, 500, 525, 550, 525, 550, 600, 625, 650, 675, 700,725, 750, or 800 contiguous amino acid residues, or up to the totalnumber of amino acid residues present in the full-length polypeptide.

Thus, in some embodiments, a variant or functional fragment of apolypeptide of this invention or a variant or functional fragment havingsubstantial identity to a polypeptide sequence of this invention (e.g.,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8) when introduced intoand expressed in a transgenic plant reduces or increases the nicotinecontent of the transgenic plant producing said polypeptides.

As used herein, the terms “express,” “expresses,” “expressed” or“expression,” and the like, with respect to a nucleic acid moleculeand/or a nucleotide sequence (e.g., RNA or DNA) indicates that thenucleic acid molecule and/or a nucleotide sequence is transcribed and,optionally, translated. Thus, a nucleic acid molecule and/or anucleotide sequence may express a polypeptide of interest or afunctional untranslated RNA.

A “heterologous” or “exogenous” nucleotide sequence is a nucleotidesequence not naturally associated with a host cell into which it isintroduced, including non-naturally occurring multiple copies of anaturally occurring nucleotide sequence. Alternatively, a heterologousnucleotide sequence can be one that does not naturally occur withanother nucleotide sequence to which it is associated. For example, anucleic acid construct comprising a “heterologous promoter” operablyassociated with a nucleic acid molecule is a promoter that does notnaturally occur with said nucleic acid molecule to which it isassociated.

A “native” or “wild type” nucleic acid, nucleotide sequence, polypeptideor amino acid sequence refers to a naturally occurring or endogenousnucleic acid, nucleotide sequence, polypeptide or amino acid sequence.Thus, for example, a “wild type mRNA” is an mRNA that is naturallyoccurring in or endogenous to the organism. A “homologous” nucleic acidsequence is a nucleotide sequence naturally associated with a host cellinto which it is introduced.

Also as used herein, the terms “nucleic acid,” “nucleic acid molecule,”“nucleotide sequence” and “polynucleotide” can be used interchangeablyand encompass both RNA and DNA, including cDNA, genomic DNA, mRNA,synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNAand DNA. The term polynucleotide, nucleotide sequence, or nucleic acidrefers to a chain of nucleotides without regard to length of the chain.The nucleic acid can be double-stranded or single-stranded. Wheresingle-stranded, the nucleic acid can be a sense strand or an antisensestrand. The nucleic acid can be synthesized using oligonucleotideanalogs or derivatives (e.g., inosine or phosphorothioate nucleotides).Such oligonucleotides can be used, for example, to prepare nucleic acidsthat have altered base-pairing abilities or increased resistance tonucleases. The present invention further provides a nucleic acid that isthe complement (which can be either a full complement or a partialcomplement) of a nucleic acid, nucleotide sequence, or polynucleotide ofthis invention. Nucleic acid molecules and/or nucleotide sequencesprovided herein are presented herein in the 5′ to 3′ direction, fromleft to right and are represented using the standard code forrepresenting the nucleotide characters as set forth in the U.S. sequencerules, 37 CFR §§ 1.821-1.825 and the World Intellectual PropertyOrganization (WIPO) Standard ST.25.

The term “antisense nucleotide sequence” or “antisense oligonucleotide”as used herein, refers to a nucleotide sequence that is complementary toa specified DNA or RNA sequence. Antisense oligonucleotides and nucleicacids that express the same can be made in accordance with conventionaltechniques. See, e.g., U.S. Pat. No. 5,023,243 to Tullis; U.S. Pat. No.5,149,797 to Pederson et al. The antisense nucleotide sequence can becomplementary to the entire nucleotide sequence encoding the polypeptideor a portion thereof of at least 10, 20, 40, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000 or more contiguous bases and will reduce the level of polypeptideproduction.

Those skilled in the art will appreciate that it is not necessary thatthe antisense nucleotide sequence be fully complementary to the targetsequence as long as the degree of sequence similarity is sufficient forthe anti sense nucleotide sequence to hybridize to its target and reduceproduction of the polypeptide or transcript. As is known in the art, ahigher degree of sequence similarity is generally required for shortantisense nucleotide sequences, whereas a greater degree of mismatchedbases will be tolerated by longer antisense nucleotide sequences.

For example, hybridization of such nucleotide sequences can be carriedout under conditions of reduced stringency, medium stringency or evenstringent conditions (e.g., conditions represented by a wash stringencyof 35-40% formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at37° C.; conditions represented by a wash stringency of 40-45% formamidewith 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and/orconditions represented by a wash stringency of 50% formamide with5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively) tothe nucleotide sequences specifically disclosed herein. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (ColdSpring Harbor, N.Y., 1989).

In other embodiments, antisense nucleotide sequences of the inventionhave at least about 70%, 80%, 90%, 95%, 97%, 98% or higher sequencesimilarity with the complement of the coding sequences specificallydisclosed herein and will reduce the level of polypeptide production.

The length of the antisense nucleotide sequence (i.e., the number ofnucleotides therein) is not critical as long as it binds selectively tothe intended location and reduces transcription and/or translation ofthe target sequence, and can be determined in accordance with routineprocedures. In general, the antisense nucleotide sequence will be fromabout eight, ten or twelve nucleotides in length to about 20, 30, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000 nucleotides, or longer, in length.

An antisense nucleotide sequence can be constructed using chemicalsynthesis and enzymatic ligation reactions by procedures known in theart. For example, an antisense nucleotide sequence can be chemicallysynthesized using naturally occurring nucleotides or various modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleotide sequences, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleotide sequence include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomet-hyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopenten-yladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleotide sequencecan be produced using an expression vector into which a nucleic acid hasbeen cloned in an antisense orientation (i.e., RNA transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest).

The antisense nucleotide sequences of the invention further includenucleotide sequences wherein at least one, or all, of theinternucleotide bridging phosphate residues are modified phosphates,such as methyl phosphonates, methyl phosphonothioates,phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. Forexample, every other one of the internucleotide bridging phosphateresidues can be modified as described. In another non-limiting example,the antisense nucleotide sequence is a nucleotide sequence in which one,or all, of the nucleotides contain a 2′ lower alkyl moiety (e.g., C₁-C₄,linear or branched, saturated or unsaturated alkyl, such as methyl,ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). Forexample, every other one of the nucleotides can be modified asdescribed. See also, Furdon et al., Nucleic Acids Res. 17:9193 (1989);Agrawal et al., Proc. Natl. Acad. Sci. USA 87:1401 (1990); Baker et al.,Nucleic Acids Res. 18:3537 (1990); Sproat et al., Nucleic Acids Res.17:3373 (1989); Walder and Walder, Proc. Natl. Acad. Sci. USA 85:5011(1988); incorporated by reference herein for their teaching of methodsof making antisense molecules, including those containing modifiednucleotide bases).

Triple helix base-pairing methods can also be employed to inhibitproduction of polypeptides of this invention (e.g., SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6 and/or SEQ ID NO:8). Triple helix pairing is believedto work by inhibiting the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (e.g., Gee et al., (1994) In: Huber etal., Molecular and Immunologic Approaches, Futura Publishing Co., Mt.Kisco, N.Y.).

Different nucleic acids or proteins having homology are referred toherein as “homologues.” The term homologue includes homologous sequencesfrom the same and other species and orthologous sequences from the sameand other species. “Homology” refers to the level of similarity betweentwo or more nucleic acid and/or amino acid sequences in terms of percentof positional identity (i.e., sequence similarity or identity). Homologyalso refers to the concept of similar functional properties amongdifferent nucleic acids or proteins. Thus, the compositions and methodsof the invention further comprise homologues to the nucleotide sequencesand polypeptide sequences of this invention. “Orthologous,” as usedherein, refers to homologous nucleotide sequences and/or amino acidsequences in different species that arose from a common ancestral geneduring speciation. A homologue of this invention has a significantsequence identity (e.g., 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or100%) to the nucleotide sequences of the invention.

As used herein “sequence identity” refers to the extent to which twooptimally aligned polynucleotide or peptide sequences are invariantthroughout a window of alignment of components, e.g., nucleotides oramino acids. “Identity” can be readily calculated by known methodsincluding, but not limited to, those described in: ComputationalMolecular Biology (Lesk, A. M., ed.) Oxford University Press, New York(1988); Biocomputing: Informatics and Genome Projects (Smith, D. W.,ed.) Academic Press, New York (1993); Computer Analysis of SequenceData, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press,New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje,G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov,M. and Devereux, J., eds.) Stockton Press, New York (1991).

As used herein, the term “percent sequence identity” or “percentidentity” refers to the percentage of identical nucleotides in a linearpolynucleotide sequence of a reference (“query”) polynucleotide molecule(or its complementary strand) as compared to a test (“subject”)polynucleotide molecule (or its complementary strand) when the twosequences are optimally aligned. In some embodiments, “percent identity”can refer to the percentage of identical amino acids in an amino acidsequence.

As used herein, the phrase “substantially identical,” in the context oftwo nucleic acid molecules, nucleotide sequences or protein sequences,refers to two or more sequences or subsequences that have at least about70%, at least about 75%, at least about 80%, at least about 81%, atleast about 82%, at least about 83%, at least about 84%, at least about85%, at least about 86%, at least about 87%, at least about 88%, atleast about 89%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. In some embodiments ofthe invention, the substantial identity exists over a region of thesequences that is at least about 50 residues to about 150 residues inlength. Thus, in some embodiments of the invention, the substantialidentity exists over a region of the sequences that is at least about50, about 60, about 70, about 80, about 90, about 100, about 110, about120, about 130, about 140, about 150, or more residues in length. Insome particular embodiments, the sequences are substantially identicalover at least about 150 residues. In a further embodiment, the sequencesare substantially identical over the entire length of the codingregions. Furthermore, in representative embodiments, substantiallyidentical nucleotide or protein sequences perform substantially the samefunction (e.g., modulating nicotine content).

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for aligning a comparison window are wellknown to those skilled in the art and may be conducted by tools such asthe local homology algorithm of Smith and Waterman, the homologyalignment algorithm of Needleman and Wunsch, the search for similaritymethod of Pearson and Lipman, and optionally by computerizedimplementations of these algorithms such as GAP, BESTFIT, FASTA, andTFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc.,San Diego, Calif.). An “identity fraction” for aligned segments of atest sequence and a reference sequence is the number of identicalcomponents which are shared by the two aligned sequences divided by thetotal number of components in the reference sequence segment, i.e., theentire reference sequence or a smaller defined part of the referencesequence. Percent sequence identity is represented as the identityfraction multiplied by 100. The comparison of one or more polynucleotidesequences may be to a full-length polynucleotide sequence or a portionthereof, or to a longer polynucleotide sequence. For purposes of thisinvention “percent identity” may also be determined using BLASTX version2.0 for translated nucleotide sequences and BLASTN version 2.0 forpolynucleotide sequences.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., 1990). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA 89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleotide sequenceto the reference nucleotide sequence is less than about 0.1 to less thanabout 0.001. Thus, in some embodiments of the invention, the smallestsum probability in a comparison of the test nucleotide sequence to thereference nucleotide sequence is less than about 0.001.

Two nucleotide sequences can also be considered to be substantiallyidentical when the two sequences hybridize to each other under stringentconditions. In some representative embodiments, two nucleotide sequencesconsidered to be substantially identical hybridize to each other underhighly stringent conditions.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. An extensiveguide to the hybridization of nucleic acids is found in TijssenLaboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes part I chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays” Elsevier, New York (1993). Generally, highlystringent hybridization and wash conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor hybridization of complementary nucleotide sequences which have morethan 100 complementary residues on a filter in a Southern or northernblot is 50% formamide with 1 mg of heparin at 42° C., with thehybridization being carried out overnight. An example of highlystringent wash conditions is 0.1 5M NaCl at 72° C. for about 15 minutes.An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for15 minutes (see, Sambrook, infra, for a description of SSC buffer).Often, a high stringency wash is preceded by a low stringency wash toremove background probe signal. An example of a medium stringency washfor a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for15 minutes. An example of a low stringency wash for a duplex of, e.g.,more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. Forshort probes (e.g., about 10 to 50 nucleotides), stringent conditionstypically involve salt concentrations of less than about 1.0 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3, and the temperature is typically at least about 30° C.Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleotide sequences that do not hybridize to each otherunder stringent conditions are still substantially identical if theproteins that they encode are substantially identical. This can occur,for example, when a copy of a nucleotide sequence is created using themaximum codon degeneracy permitted by the genetic code.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone homologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of theinvention. In one embodiment, a reference nucleotide sequence hybridizesto the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS),0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50°C. In another embodiment, the reference nucleotide sequence hybridizesto the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS),0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50°C. or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C. In still furtherembodiments, the reference nucleotide sequence hybridizes to the “test”nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., or in 7%sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. withwashing in 0.1×SSC, 0.1% SDS at 65° C.

In particular embodiments, a further indication that two nucleotidesequences or two polypeptide sequences are substantially identical canbe that the protein encoded by the first nucleic acid is immunologicallycross reactive with, or specifically binds to, the protein encoded bythe second nucleic acid. Thus, in some embodiments, a polypeptide can besubstantially identical to a second polypeptide, for example, where thetwo polypeptides differ only by conservative substitutions.

An “isolated” nucleic acid molecule, an “isolated” nucleotide sequenceor an “isolated” polypeptide is a nucleic acid molecule, nucleotidesequence or polypeptide that, by the hand of man, exists apart from itsnative environment and is therefore not a product of nature. An isolatednucleic acid molecule, nucleotide sequence or polypeptide may exist in apurified form that is at least partially separated from at least some ofthe other components of the naturally occurring organism or virus, forexample, the cell or viral structural components or other polypeptidesor nucleic acids commonly found associated with the polynucleotide. Inrepresentative embodiments, the isolated nucleic acid molecule, theisolated nucleotide sequence and/or the isolated polypeptide is at leastabout 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or morepure.

In other embodiments, an isolated nucleic acid molecule, nucleotidesequence or polypeptide may exist in a non-native environment such as,for example, a recombinant host cell. Thus, for example, with respect tonucleotide sequences, the term “isolated” means that it is separatedfrom the chromosome and/or cell in which it naturally occurs. Apolynucleotide is also isolated if it is separated from the chromosomeand/or cell in which it naturally occurs in and is then inserted into agenetic context, a chromosome and/or a cell in which it does notnaturally occur (e.g., a different host cell, different regulatorysequences, and/or different position in the genome than as found innature). Accordingly, the recombinant nucleic acid molecules, nucleotidesequences and their encoded polypeptides are “isolated” in that, by thehand of man, they exist apart from their native environment andtherefore are not products of nature, however, in some embodiments, theycan be introduced into and exist in a recombinant host cell.

In some embodiments, the nucleotide sequences and/or nucleic acidmolecules of the invention can be operatively associated with a varietyof promoters for expression in host cells (e.g., plant cells). Thus, insome embodiments, the invention provides transformed host cells andtransformed organisms comprising the transformed host cells, wherein thehost cells and organisms are transformed with one or more nucleic acidmolecules/nucleotide sequences of the invention. As used herein,“operatively associated with,” when referring to a first nucleic acidsequence that is operatively linked to a second nucleic acid sequence,means a situation when the first nucleic acid sequence is placed in afunctional relationship with the second nucleic acid sequence. Forinstance, a promoter is operatively associated with a coding sequence ifthe promoter effects the transcription or expression of the codingsequence.

A promoter can be any promoter useful for expression of nucleic acids inplants and as described herein. In some embodiments, the promoter can bea constitutive promoter. In other embodiments, it can be a tissuepreferred promoter or a tissue specific promoter.

A DNA “promoter” is an untranslated DNA sequence upstream of a codingregion that contains the binding site for RNA polymerase and initiatestranscription of the DNA. A “promoter region” can also include otherelements that act as regulators of gene expression. Promoters caninclude, for example, constitutive, inducible, temporally regulated,developmentally regulated, chemically regulated, tissue-preferred andtissue-specific promoters for use in the preparation of recombinantnucleic acid molecules, i.e., chimeric genes. In particular aspects, a“promoter” useful with the invention is a promoter capable of initiatingtranscription of a nucleotide sequence in a cell of a plant.

The choice of promoter will vary depending on the temporal and spatialrequirements for expression, and also depending on the host cell to betransformed. Thus, for example, expression of the nucleotide sequencesof the invention can be in any plant and/or plant part, (e.g., inleaves, in stalks or stems, in ears, in inflorescences, in roots, seedsand/or seedlings, and the like).

Promoters useful with the invention include, but are not limited to,those that drive expression of a nucleotide sequence constitutively,those that drive expression when induced, and those that driveexpression in a tissue- or developmentally-specific manner. Thesevarious types of promoters are known in the art.

Examples of constitutive promoters include, but are not limited to,cestrum virus promoter (cmp) (U.S. Pat. No. 7,166,770), the rice actin 1promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; as well asU.S. Pat. No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol.9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci USA84:5745-5749), Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci.USA 84:6624-6629), sucrose synthase promoter (Yang & Russell (1990)Proc. Natl. Acad. Sci. USA 87:4144-4148), and the ubiquitin promoter.The constitutive promoter derived from ubiquitin accumulates in manycell types. Ubiquitin promoters have been cloned from several plantspecies for use in transgenic plants, for example, sunflower (Binet etal., 1991. Plant Science 79: 87-94), maize (Christensen et al., 1989.Plant Molec. Biol. 12: 619-632), and Arabidopsis (Norris et al. 1993.Plant Molec. Biol. 21:895-906). The maize ubiquitin promoter (UbiP) hasbeen developed in transgenic monocot systems and its sequence andvectors constructed for monocot transformation are disclosed in thepatent publication EP 0 342 926. The ubiquitin promoter is suitable forthe expression of the nucleotide sequences of the invention intransgenic plants, especially monocotyledons. Further, the promoterexpression cassettes described by McElroy et al. (Mol. Gen. Genet. 231:150-160 (1991)) can be easily modified for the expression of thenucleotide sequences of the invention and are particularly suitable foruse in monocotyledonous hosts.

In some embodiments, tissue specific/tissue preferred promoters can beused. Tissue specific or preferred expression patterns include, but arenot limited to, green tissue specific or preferred, root specific orpreferred, stem specific or preferred, and flower specific or preferred.Promoters suitable for expression in green tissue include many thatregulate genes involved in photosynthesis and many of these have beencloned from both monocotyledons and dicotyledons. In one embodiment, apromoter useful with the invention is the maize PEPC promoter from thephosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol.12:579-589 (1989)). Non-limiting examples of tissue-specific promotersinclude those associated with genes encoding the seed storage proteins(such as β-conglycinin, cruciferin, napin and phaseolin), zein or oilbody proteins (such as oleosin), or proteins involved in fatty acidbiosynthesis (including acyl carrier protein, stearoyl-ACP desaturaseand fatty acid desaturases (fad 2-1)), and other nucleic acids expressedduring embryo development (such as Bce4, see, e.g., Kridl et al. (1991)Seed Sci. Res. 1:209-219; as well as EP Patent No. 255378).Tissue-specific or tissue-preferential promoters useful for theexpression of the nucleotide sequences of the invention in plants,particularly maize, include but are not limited to those that directexpression in root, pith, leaf or pollen. Such promoters are disclosed,for example, in WO 93/07278, herein incorporated by reference in itsentirety. Other non-limiting examples of tissue specific or tissuepreferred promoters useful with the invention the cotton rubiscopromoter disclosed in U.S. Pat. No. 6,040,504; the rice sucrose synthasepromoter disclosed in U.S. Pat. No. 5,604,121; the root specificpromoter described by de Framond (FEBS 290:103-106 (1991); EP 0 452 269to Ciba-Geigy); the stem specific promoter described in U.S. Pat. No.5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpAgene; and the cestrum yellow leaf curling virus promoter disclosed in WO01/73087, all incorporated by reference

Additional examples of tissue-specific/tissue preferred promotersinclude, but are not limited to, the root-specific promoters RCc3 (Jeonget al. Plant Physiol. 153:185-197 (2010)) and RB7 (U.S. Pat. No.5,459,252), the lectin promoter (Lindstrom et al. (1990) Der. Genet.11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), cornalcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic AcidsRes. 12:3983-4000), S-adenosyl-L-methionine synthetase (SAMS) (VanderMijnsbrugge et al. (1996) Plant and Cell Physiology, 37(8):1108-1115),corn light harvesting complex promoter (Bansal et al. (1992) Proc. Natl.Acad. Sci. USA 89:3654-3658), corn heat shock protein promoter (O'Dellet al. (1985) EMBO J. 5:451-458; and Rochester et al. (1986) EMBO J.5:451-458), pea small subunit RuBP carboxylase promoter (Cashmore,“Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphatecarboxylase” pp. 29-39 In: Genetic Engineering of Plants (Hollaendered., Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet.205:193-200), Ti plasmid mannopine synthase promoter (Langridge et al.(1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), Ti plasmid nopalinesynthase promoter (Langridge et al. (1989), supra), petunia chalconeisomerase promoter (van Tunen et al. (1988) EMBO J. 7:1257-1263), beanglycine rich protein 1 promoter (Keller et al. (1989) Genes Dev.3:1639-1646), truncated CaMV 35S promoter (O'Dell et al. (1985) Nature313:810-812), potato patatin promoter (Wenzler et al. (1989) Plant Mol.Biol. 13:347-354), root cell promoter (Yamamoto et al. (1990) NucleicAcids Res. 18:7449), maize zein promoter (Kriz et al. (1987) Mol. Gen.Genet. 207:90-98; Langridge et al. (1983) Cell 34:1015-1022; Reina etal. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic AcidsRes. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354),globulin-1 promoter (Belanger et al. (1991) Genetics 129:863-872),α-tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet.215:431-440), PEPCase promoter (Hudspeth & Grula (1989) Plant Mol. Biol.12:579-589), R gene complex-associated promoters (Chandler et al. (1989)Plant Cell 1:1175-1183), and chalcone synthase promoters (Franken et al.(1991) EMBO J. 10:2605-2612). In some particular embodiments, thenucleotide sequences of the invention are operatively associated with aroot-preferred promoter.

Particularly useful for seed-specific expression is the pea vicilinpromoter (Czako et al. (1992) Mol. Gen. Genet. 235:33-40; as well as theseed-specific promoters disclosed in U.S. Pat. No. 5,625,136. Usefulpromoters for expression in mature leaves are those that are switched onat the onset of senescence, such as the SAG promoter from Arabidopsis(Gan et al. (1995) Science 270:1986-1988).

In addition, promoters functional in plastids can be used. Non-limitingexamples of such promoters include the bacteriophage T3 gene 9 5′ UTRand other promoters disclosed in U.S. Pat. No. 7,579,516. Otherpromoters useful with the invention include but are not limited to theS-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsininhibitor gene promoter (Kti3).

In some embodiments of the invention, inducible promoters can be used.Thus, for example, chemical-regulated promoters can be used to modulatethe expression of a gene in a plant through the application of anexogenous chemical regulator. Regulation of the expression of nucleotidesequences of the invention via promoters that are chemically regulatedenables the polypeptides of the invention to be synthesized only whenthe crop plants are treated with the inducing chemicals. Depending uponthe objective, the promoter may be a chemical-inducible promoter, whereapplication of a chemical induces gene expression, or achemical-repressible promoter, where application of the chemicalrepresses gene expression.

Chemical inducible promoters are known in the art and include, but arenot limited to, the maize In2-2 promoter, which is activated bybenzenesulfonamide herbicide safeners, the maize GST promoter, which isactivated by hydrophobic electrophilic compounds that are used aspre-emergent herbicides, and the tobacco PR-1 a promoter, which isactivated by salicylic acid (e.g., the PR1a system), steroidsteroid-responsive promoters (see, e.g., the glucocorticoid-induciblepromoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88,10421-10425 and McNellis et al. (1998) Plant J. 14, 247-257) andtetracycline-inducible and tetracycline-repressible promoters (see,e.g., Gatz et al. (1991) Mol. Gen. Genet. 227, 229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, Lac repressor system promoters,copper-inducible system promoters, salicylate-inducible system promoters(e.g., the PR1a system), glucocorticoid-inducible promoters (Aoyama etal. (1997) Plant J. 11:605-612), and ecdysone-inducible systempromoters.

Other non-limiting examples of inducible promoters include ABA- andturgor-inducible promoters, the auxin-binding protein gene promoter(Schwob et al. (1993) Plant J. 4:423-432), the UDP glucose flavonoidglycosyl-transferase promoter (Ralston et al. (1988) Genetics119:185-197), the MPI proteinase inhibitor promoter (Cordero et al.(1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphatedehydrogenase promoter (Kohler et al. (1995) Plant Mol. Biol.29:1293-1298; Martinez et al. (1989) J. Mol. Biol. 208:551-565; andQuigley et al. (1989) J. Mol. Evol. 29:412-421). Also included are thebenzene sulphonamide-inducible (U.S. Pat. No. 5,364,780) andalcohol-inducible (Int'l Patent Application Publication Nos. WO 97/06269and WO 97/06268) systems and glutathione S-transferase promoters.Likewise, one can use any of the inducible promoters described in Gatz(1996) Current Opinion Biotechnol. 7:168-172 and Gatz (1997) Annu. Rev.Plant Physiol. Plant Mol. Biol. 48:89-108. Other chemically induciblepromoters useful for directing the expression of the nucleotidesequences of this invention in plants are disclosed in U.S. Pat. No.5,614,395 herein incorporated by reference in its entirety. Chemicalinduction of gene expression is also detailed in the publishedapplication EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395. Insome embodiments, a promoter for chemical induction can be the tobaccoPR-1a promoter.

In further aspects, the nucleotide sequences of the invention can beoperatively associated with a promoter that is wound inducible orinducible by pest or pathogen infection. Numerous promoters have beendescribed which are expressed at wound sites and/or at the sites of pestattack or phytopathogen infection. Ideally, such a promoter should beactive only locally at or adjacent to the sites of attack, and in thisway expression of the nucleotide sequences of the invention will befocused in the cells that are being invaded. Such promoters include, butare not limited to, those described by Stanford et al., Mol. Gen. Genet.215:200-208 (1989), Xu et al. Plant Molec. Biol. 22:573-588 (1993),Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier and Lehle, PlantMolec. Biol. 22:783-792 (1993), Firek et al. Plant Molec. Biol.22:129-142 (1993), Warner et al. Plant J. 3:191-201 (1993), U.S. Pat.Nos. 5,750,386, 5,955,646, 6,262,344, 6,395,963, 6,703,541, 7,078,589,7,196,247, 7,223,901, and U.S. Patent Application Publication2010043102.

As used herein, “expression cassette” means a nucleic acid constructcomprising a nucleotide sequence of interest (e.g., the nucleotidesequences of the invention), wherein said nucleotide sequence isoperatively associated with at least a control sequence (e.g., apromoter). Thus, some embodiments of the invention provide expressioncassettes designed to express the nucleotides sequences of theinvention. In this manner, for example, one or more plant promotersoperatively associated with one or more nucleotide sequences of theinvention (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and/or SEQ IDNO:7) are provided in expression cassettes for expression in an organismor cell thereof (e.g., a plant, plant part and/or plant cell).

An expression cassette comprising a nucleotide sequence of interest maybe chimeric, meaning that at least one of its components is heterologouswith respect to at least one of its other components. An expressioncassette may also be one that is naturally occurring but has beenobtained in a recombinant form useful for heterologous expression.Typically, however, the expression cassette is heterologous with respectto the host, i.e., the particular nucleic acid sequence of theexpression cassette does not occur naturally in the host cell and musthave been introduced into the host cell or an ancestor of the host cellby a transformation event.

In addition to the promoters operatively linked to the nucleotidesequences of the invention, an expression cassette of the invention canalso include other regulatory sequences. As used herein, “regulatorysequences” means nucleotide sequences located upstream (5′ non-codingsequences), within or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences include, but are not limited to, promoters, enhancers,introns, translation leader sequences, termination signals, andpolyadenylation signal sequences.

For purposes of the invention, the regulatory sequences or regions canbe native/analogous to the plant, plant part and/or plant cell and/orthe regulatory sequences can be native/analogous to the other regulatorysequences. Alternatively, the regulatory sequences may be heterologousto the plant (and/or plant part and/or plant cell) and/or to each other(i.e., the regulatory sequences). Thus, for example, a promoter can beheterologous when it is operatively linked to a polynucleotide sequencefrom a species different from the species from which the polynucleotidesequence was derived. Alternatively, a promoter can also be heterologousto a selected nucleotide sequence if the promoter is from thesame/analogous species from which the polynucleotide is derived, but oneor both (i.e., promoter and/or polynucleotide) are substantiallymodified from their original form and/or genomic locus, and/or thepromoter is not the native promoter for the operably linkedpolynucleotide.

A number of non-translated leader sequences derived from viruses areknown to enhance gene expression. Specifically, leader sequences fromTobacco Mosaic Virus (TMV, the “ω-sequence”), Maize Chlorotic MottleVirus (MCMV) and Alfalfa Mosaic Virus (AMV) have been shown to beeffective in enhancing expression (Gallie et al. (1987) Nucleic AcidsRes. 15:8693-8711; and Skuzeski et al. (1990) Plant Mol. Biol.15:65-79). Other leader sequences known in the art include, but are notlimited to, picornavirus leaders such as an encephalomyocarditis (EMCV)5′ noncoding region leader (Elroy-Stein et al. (1989) Proc. Natl. Acad.Sci. USA 86:6126-6130); potyvirus leaders such as a Tobacco Etch Virus(TEV) leader (Allison et al. (1986) Virology 154:9-20); Maize DwarfMosaic Virus (MDMV) leader (Allison et al. (1986), supra); humanimmunoglobulin heavy-chain binding protein (BiP) leader (Macejak & Samow(1991) Nature 353:90-94); untranslated leader from the coat protein mRNAof AMV (AMV RNA 4; Jobling & Gehrke (1987) Nature 325:622-625); tobaccomosaic TMV leader (Gallie et al. (1989) Molecular Biology of RNA237-256); and MCMV leader (Lommel et al. (1991) Virology 81:382-385).See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

An expression cassette also can optionally include a transcriptionaland/or translational termination region (i.e., termination region) thatis functional in plants. A variety of transcriptional terminators areavailable for use in expression cassettes and are responsible for thetermination of transcription beyond the heterologous nucleotide sequenceof interest and correct mRNA polyadenylation. The termination region maybe native to the transcriptional initiation region, may be native to theoperably linked nucleotide sequence of interest, may be native to theplant host, or may be derived from another source (i.e., foreign orheterologous to the promoter, the nucleotide sequence of interest, theplant host, or any combination thereof). Appropriate transcriptionalterminators include, but are not limited to, the CAMV 35S terminator,the tml terminator, the nopaline synthase terminator and/or the pea rbcsE9 terminator. These can be used in both monocotyledons anddicotyledons. In addition, a coding sequence's native transcriptionterminator can be used.

An expression cassette of the invention also can include a nucleotidesequence for a selectable marker, which can be used to select atransformed plant, plant part and/or plant cell. As used herein,“selectable marker” means a nucleotide sequence that when expressedimparts a distinct phenotype to the plant, plant part and/or plant cellexpressing the marker and thus allows such transformed plants, plantparts and/or plant cells to be distinguished from those that do not havethe marker. Such a nucleotide sequence may encode either a selectable orscreenable marker, depending on whether the marker confers a trait thatcan be selected for by chemical means, such as by using a selectiveagent (e.g., an antibiotic, herbicide, or the like), or if the marker issimply a trait that one can identify through observation or testing,such as by screening (e.g., the R-locus trait). Of course, many examplesof suitable selectable markers are known in the art and can be used inthe expression cassettes described herein.

Examples of selectable markers include, but are not limited to, anucleotide sequence encoding neo or nptII, which confers resistance tokanamycin, G418, and the like (Potrykus et al. (1985) Mol. Gen. Genet.199:183-188); a nucleotide sequence encoding bar, which confersresistance to phosphinothricin; a nucleotide sequence encoding analtered 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, whichconfers resistance to glyphosate (Hinchee et al. (1988) Biotech.6:915-922); a nucleotide sequence encoding a nitrilase such as bxn fromKlebsiella ozaenae that confers resistance to bromoxynil (Stalker et al.(1988) Science 242:419-423); a nucleotide sequence encoding an alteredacetolactate synthase (ALS) that confers resistance to imidazolinone,sulfonylurea or other ALS-inhibiting chemicals (EP Patent ApplicationNo. 154204); a nucleotide sequence encoding a methotrexate-resistantdihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem.263:12500-12508); a nucleotide sequence encoding a dalapon dehalogenasethat confers resistance to dalapon; a nucleotide sequence encoding amannose-6-phosphate isomerase (also referred to as phosphomannoseisomerase (PMI)) that confers an ability to metabolize mannose (U.S.Pat. Nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding analtered anthranilate synthase that confers resistance to 5-methyltryptophan; and/or a nucleotide sequence encoding hph that confersresistance to hygromycin. One of skill in the art is capable of choosinga suitable selectable marker for use in an expression cassette of theinvention.

Additional selectable markers include, but are not limited to, anucleotide sequence encoding β-glucuronidase or uidA (GUS) that encodesan enzyme for which various chromogenic substrates are known; an R-locusnucleotide sequence that encodes a product that regulates the productionof anthocyanin pigments (red color) in plant tissues (Dellaporta et al.,“Molecular cloning of the maize R-nj allele by transposon-tagging withAc,” pp. 263-282 In: Chromosome Structure and Function: Impact of NewConcepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds.,Plenum Press 1988)); a nucleotide sequence encoding β-lactamase, anenzyme for which various chromogenic substrates are known (e.g., PADAC,a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci.USA 75:3737-3741); a nucleotide sequence encoding xylE that encodes acatechol dioxygenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone, which in turncondenses to form melanin (Katz et al. (1983) J. Gen. Microbiol.129:2703-2714); a nucleotide sequence encoding β-galactosidase, anenzyme for which there are chromogenic substrates; a nucleotide sequenceencoding luciferase (lux) that allows for bioluminescence detection (Owet al. (1986) Science 234:856-859); a nucleotide sequence encodingaequorin, which may be employed in calcium-sensitive bioluminescencedetection (Prasher et al. (1985) Biochem. Biophys. Res. Comm.126:1259-1268); or a nucleotide sequence encoding green fluorescentprotein (Niedz et al. (1995) Plant Cell Reports 14:403-406). One ofskill in the art is capable of choosing a suitable selectable marker foruse in an expression cassette of the invention.

An expression cassette of the invention also can include nucleotidesequences that encode other desired traits. Such desired traits can beother nucleotide sequences which confer various agriculturally desirabletraits such as disease and/or insect resistance, abiotic stresstolerance or resistance and the like. Such nucleotide sequences can bestacked with any combination of nucleotide sequences to create plants,plant parts or plant cells having the desired phenotype. Stackedcombinations can be created by any method including, but not limited to,cross breeding plants by any conventional methodology, or by genetictransformation. If stacked by genetically transforming the plants,nucleotide sequences encoding additional desired traits can be combinedat any time and in any order. For example, a transgenic plant comprisingone or more desired traits can be used as the target to introducefurther traits by subsequent transformation. The additional nucleotidesequences can be introduced simultaneously in a co-transformationprotocol with a nucleotide sequence, nucleic acid molecule, nucleic acidconstruct, and/or composition of the invention, provided by anycombination of expression cassettes. For example, if two nucleotidesequences will be introduced, they can be incorporated in separatecassettes (trans) or can be incorporated on the same cassette (cis).Expression of the nucleotide sequences can be driven by the samepromoter or by different promoters. It is further recognized thatnucleotide sequences can be stacked at a desired genomic location usinga site-specific recombination system. See, e.g., Int'l PatentApplication Publication Nos. WO 99/25821; WO 99/25854; WO 99/25840; WO99/25855 and WO 99/25853.

In addition to expression cassettes, the nucleic acid molecules andnucleotide sequences described herein can be used in connection withvectors. The term “vector” refers to a composition for transferring,delivering or introducing a nucleic acid (or nucleic acids) into a cell.A vector comprises a nucleic acid molecule comprising the nucleotidesequence(s) to be transferred, delivered or introduced. Vectors for usein transformation of plants and other organisms are well known in theart. Non-limiting examples of general classes of vectors include, butare not limited to, a viral vector, a plasmid vector, a phage vector, aphagemid vector, a cosmid, a fosmid, a bacteriophage, or an artificialchromosome. The selection of a vector will depend upon the preferredtransformation technique and the target species for transformation.Accordingly, in further embodiments, a recombinant nucleic acid moleculeof the invention can be comprised within a recombinant vector. The sizeof a vector can vary considerably depending on whether the vectorcomprises one or multiple expression cassettes (e.g., for molecularstacking). Thus, a vector size can range from about 3 kb to about 30 kb.Thus, in some embodiments, a vector is about 3 kb, 4 kb, 5 kb, 6 kb, 7kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb,18 kb, 19 kb, 20 kb, 21 kb, 22 kb, 23 kb, 24 kb, 25 kb, 26 kb, 27 kb, 28kb, 29 kb, 30 kb, 40 kb, 50 kb, 60 kb, and the like or any rangetherein, in size. In some particular embodiments, a vector can be about3 kb to about 10 kb in size.

The present invention is directed in part to the discovery thatmodulating expression by over expressing or inhibiting expression in aplant of at least one isolated nucleic acid molecule or nucleic acidconstruct of this invention can result in the plant having increasednicotine or reduced nicotine content as compared to a plant that doesnot comprise said isolate nucleic acid molecule or nucleic acidconstruct.

Thus, in some embodiments of the invention, a method of producing atransgenic plant cell is provided, said method comprising introducinginto a plant cell an isolated nucleic acid molecule/construct of theinvention, thereby producing a transgenic plant cell that can regeneratea transgenic plant having modulated (e.g., increased or decreased)alkaloid (e.g., nornicotine, nicotine, anabasine, anatabine, and thelike) content as compared to a plant regenerated from a plant cell thatdoes not comprise said nucleic acid molecule/construct. In someembodiments, the transgenic plant cell comprises more than one (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, etc.) nucleic acid molecule/nucleotide sequenceof the invention. Thus, in some aspects of the invention, the transgenicplants, or parts thereof, comprise and express one or more isolatednucleic acid molecule/constructs of the invention, thereby producing oneor more polypeptides of the invention resulting in modulated (e.g.,reduced or increased) alkaloid content in said transgenic plant.

In further embodiments of the invention, a method of producing atransgenic plant cell is provided, said method comprising introducinginto a plant cell an isolated nucleic acid molecule/construct of theinvention, thereby producing a transgenic plant cell that can regeneratea transgenic plant having modulated (e.g., increased or decreased)nicotine content as compared to a plant regenerated from a plant cellthat does not comprise said nucleic acid molecule/construct. In someembodiments, the transgenic plant cell comprises more than one (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, etc.) nucleic acid molecule/nucleotide sequenceof the invention. Thus, in some aspects of the invention, the transgenicplants, or parts thereof, comprise and express one or more isolatednucleic acid molecule/constructs of the invention, thereby producing oneor more polypeptides of the invention resulting in modulated (e.g.,reduced or increased) nicotine content in said transgenic plant.

“Introducing,” in the context of a nucleotide sequence of interest(e.g., the nucleic acid molecules/constructs of the invention), meanspresenting the nucleotide sequence of interest to the plant, plant part,and/or plant cell in such a manner that the nucleotide sequence gainsaccess to the interior of a cell. Where more than one nucleotidesequence is to be introduced these nucleotide sequences can be assembledas part of a single polynucleotide or nucleic acid construct, or asseparate polynucleotide or nucleic acid constructs, and can be locatedon the same or different transformation vectors. Accordingly, thesepolynucleotides can be introduced into plant cells in a singletransformation event, in separate transformation events, or, e.g., aspart of a breeding protocol. Thus, the term “transformation” as usedherein refers to the introduction of a heterologous nucleic acid into acell. Transformation of a cell may be stable or transient. Thus, in someembodiments, a plant cell of the invention is stably transformed with anucleic acid molecule of the invention. In other embodiments, a plant ofthe invention is transiently transformed with a nucleic acid molecule ofthe invention.

“Transient transformation” in the context of a polynucleotide means thata polynucleotide is introduced into the cell and does not integrate intothe genome or plastome of the cell and consequently said cell cannot beregenerated into a stably transformed plant.

By “stably introducing” or “stably introduced” in the context of apolynucleotide introduced into a cell is intended the introducedpolynucleotide is stably incorporated into the genome of the cell, andthus the cell is stably transformed with the polynucleotide.

“Stable transformation” or “stably transformed” as used herein meansthat a nucleic acid is introduced into a cell and integrates into thegenome of the cell. As such, the integrated nucleic acid is capable ofbeing inherited by the progeny thereof, more particularly, by theprogeny of multiple successive generations. “Genome” as used herein alsoincludes the nuclear and the plastid genome, and therefore includesintegration of the nucleic acid into, for example, the chloroplastgenome. Stable transformation as used herein can also refer to atransgene that is maintained extrachromasomally, for example, as aminichromosome.

Transient transformation may be detected by, for example, anenzyme-linked immunosorbent assay (ELISA) or Western blot, which candetect the presence of a peptide or polypeptide encoded by one or moretransgene introduced into an organism. Stable transformation of a cellcan be detected by, for example, a Southern blot hybridization assay ofgenomic DNA of the cell with nucleic acid sequences which specificallyhybridize with a nucleotide sequence of a transgene introduced into anorganism (e.g., a plant). Stable transformation of a cell can bedetected by, for example, a Northern blot hybridization assay of RNA ofthe cell with nucleic acid sequences which specifically hybridize with anucleotide sequence of a transgene introduced into a plant or otherorganism. Stable transformation of a cell can also be detected by, e.g.,a polymerase chain reaction (PCR) or other amplification reactions asare well known in the art, employing specific primer sequences thathybridize with target sequence(s) of a transgene, resulting inamplification of the transgene sequence, which can be detected accordingto standard methods Transformation can also be detected by directsequencing and/or hybridization protocols well known in the art.

A nucleic acid molecule of the invention (e.g., one or more of thenucleotide sequences of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO:5, SEQ IDNO:7, and/or a nucleotide sequence encoding one or more polypeptideshaving the amino acid sequence of any one of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8) can be introduced into a cell by any methodknown to those of skill in the art.

In some embodiments of the invention, transformation of a cell comprisesnuclear transformation. In other embodiments, transformation of a cellcomprises plastid transformation (e.g., chloroplast transformation).

Procedures for transforming plants are well known and routine in the artand are described throughout the literature. Non-limiting examples ofmethods for transformation of plants include transformation viabacterial-mediated nucleic acid delivery (e.g., via Agrobacteria),viral-mediated nucleic acid delivery, silicon carbide or nucleic acidwhisker-mediated nucleic acid delivery, liposome mediated nucleic aciddelivery, microinjection, microparticle bombardment,calcium-phosphate-mediated transformation, cyclodextrin-mediatedtransformation, electroporation, nanoparticle-mediated transformation,sonication, infiltration, PEG-mediated nucleic acid uptake, as well asany other electrical, chemical, physical (mechanical) and/or biologicalmechanism that results in the introduction of nucleic acid into theplant cell, including any combination thereof. General guides to variousplant transformation methods known in the art include Miki et al.(“Procedures for Introducing Foreign DNA into Plants” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) andRakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

Agrobacterium-mediated transformation is a commonly used method fortransforming plants, in particular, dicot plants, because of its highefficiency of transformation and because of its broad utility with manydifferent species. Agrobacterium-mediated transformation typicallyinvolves transfer of the binary vector carrying the foreign DNA ofinterest to an appropriate Agrobacterium strain that may depend on thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (Uknes et al. (1993) PlantCell 5:159-169). The transfer of the recombinant binary vector toAgrobacterium can be accomplished by a triparental mating procedureusing Escherichia coli carrying the recombinant binary vector, a helperE. coli strain that carries a plasmid that is able to mobilize therecombinant binary vector to the target Agrobacterium strain.Alternatively, the recombinant binary vector can be transferred toAgrobacterium by nucleic acid transformation (Höfgen & Willmitzer (1988)Nucleic Acids Res. 16:9877).

Transformation of a plant by recombinant Agrobacterium usually involvesco-cultivation of the Agrobacterium with explants from the plant andfollows methods well known in the art. Transformed tissue is regeneratedon selection medium carrying an antibiotic or herbicide resistancemarker between the binary plasmid T-DNA borders.

Another method for transforming plants, plant parts and/or plant cellsinvolves propelling inert or biologically active particles at planttissues and cells. See, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006 and5,100,792. Generally, this method involves propelling inert orbiologically active particles at the plant cells under conditionseffective to penetrate the outer surface of the cell and affordincorporation within the interior thereof. When inert particles areutilized, the vector can be introduced into the cell by coating theparticles with the vector containing the nucleic acid of interest.Alternatively, a cell or cells can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried yeast cells, dried bacteriumor a bacteriophage, each containing one or more nucleic acids sought tobe introduced) also can be propelled into plant tissue.

Thus, in particular embodiments of the invention, a plant cell can betransformed by any method known in the art and as described herein andintact plants can be regenerated from these transformed cells using anyof a variety of known techniques. Plant regeneration from plant cells,plant tissue culture and/or cultured protoplasts is described, forexample, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1,MacMillan Publishing Co. New York (1983)); and Vasil I. R. (ed.) (CellCulture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol.I (1984), and Vol. II (1986)). Methods of selecting for transformedtransgenic plants, plant cells and/or plant tissue culture are routinein the art and can be employed in the methods of the invention providedherein.

Likewise, the genetic properties engineered into the transgenic seedsand plants, plant parts, and/or plant cells of the invention describedabove can be passed on by sexual reproduction or vegetative growth andtherefore can be maintained and propagated in progeny plants. Generally,maintenance and propagation make use of known agricultural methodsdeveloped to fit specific purposes such as harvesting, sowing ortilling.

A nucleotide sequence therefore can be introduced into the plant, plantpart and/or plant cell in any number of ways that are well known in theart. The methods of the invention do not depend on a particular methodfor introducing one or more nucleotide sequences into a plant, only thatthey gain access to the interior of at least one cell of the plant.Where more than one nucleotide sequence is to be introduced, they can beassembled as part of a single nucleic acid construct, or as separatenucleic acid constructs, and can be located on the same or differentnucleic acid constructs. Accordingly, the nucleotide sequences can beintroduced into the cell of interest in a single transformation event,in separate transformation events, or, for example, in plants, as partof a breeding protocol.

Thus, in additional embodiments, the invention provides a method ofproducing a plant having a plant having modulated alkaloid content isprovided, the method comprising introducing into a plant cell a nucleicacid construct of the invention to produce a transgenic plant cell,wherein the transgenic plant cell comprises said nucleic acid constructof the invention in its genome; and regenerating said transgenic plantcell to produce a transgenic plant comprising said nucleic acidconstruct, thereby producing a plant having modulated alkaloid content.In some embodiments, the alkaloid content of the transgenic plant isincreased as compared to a plant that does not comprise said nucleicacid construct. In other embodiments, the alkaloid content of thetransgenic plant is decreased as compared to a plant that does notcomprise said nucleic acid construct. In representative embodiments, theplant is a tobacco plant.

In further embodiments, the invention provides a method of producing aplant having modulated nicotine content is provided, the methodcomprising introducing into a plant cell a nucleic acid construct of theinvention to produce a transgenic plant cell, wherein the transgenicplant cell comprises said nucleic acid construct of the invention in itsgenome; and regenerating said transgenic plant cell to produce atransgenic plant comprising said nucleic acid construct, therebyproducing a plant having modulated nicotine content. In someembodiments, the nicotine content of the transgenic plant is increasedas compared to a plant that does not comprise said nucleic acidconstruct. In other embodiments, the nicotine content of the transgenicplant is decreased as compared to a plant that does not comprise saidnucleic acid construct. In representative embodiments, the plant is atobacco plant.

In a further embodiment, the present invention provides a method ofmodulating alkaloid content in a plant, comprising introducing into aplant cell a nucleic acid construct of the invention to produce atransgenic plant cell comprising said nucleic acid construct; andregenerating said transgenic plant cell to produce a transgenic plantcomprising said nucleic acid construct, thereby modulating alkaloidcontent in said transgenic plant as compared to a plant that is nottransformed with the said nucleic acid construct. In some embodiments,the alkaloid content of the transgenic plant is increased as compared toa plant that does not comprise said nucleic acid construct. In otherembodiments, the alkaloid content of the transgenic plant is decreasedas compared to a plant that does not comprise said nucleic acidconstruct. In representative embodiments, the plant is a tobacco plant.

In a further embodiment, the present invention provides a method ofmodulating nicotine content in a plant, comprising introducing into aplant cell a nucleic acid construct of the invention to produce atransgenic plant cell comprising said nucleic acid construct; andregenerating said transgenic plant cell to produce a transgenic plantcomprising said nucleic acid construct, thereby modulating nicotineproduction in said transgenic plant as compared to a plant that is nottransformed with the (does not comprise) said nucleic acid construct. Insome embodiments, the nicotine content of the transgenic plant isincreased as compared to a plant that does not comprise said nucleicacid construct. In other embodiments, the nicotine content of thetransgenic plant is decreased as compared to a plant that does notcomprise said nucleic acid construct. In representative embodiments, theplant is a tobacco plant.

The present invention further provides methods of modulating alkaloid(e.g., nornicotine, nicotine, anabasine, anatabine, and the like)content in a plant and methods of producing plants having modulatedalkaloid (e.g., nornicotine, nicotine, anabasine, anatabine, and thelike) content comprising in planta modification of one or more of thewild-type or native nucleotide sequences encoding the transcriptionfactors of this invention (e.g., NtMYC2a, NtMYC2b, NtERF98, NtETTa). Anymethod of modify a nucleotide sequence in planta can be used with thisinvention to alter the expression of the genes encoding thesetranscription factors. Such methods can include, but are not limited to,mutagenesis or gene targeting/editing using meganuclease, Zinc Fingernuclease, TALENs, and/or CRISPR/Cas9 nuclease and/or introduction of anucleic acid or gene repair oligonucleobase comprising at least aportion of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7. In someembodiments, the nucleotide sequence of a transcription factor of theinvention can be modified such that an amino acid codon is substitutedfor a stop codon, resulting in premature termination during translationof the polypeptide and thus resulting in reduced or no activity of thetranscription factor, thereby modulating the alkaloid content of theplant.

Procedures for determining nicotine and alkaloid content are well knownand routine in the art and are described throughout the literature.Non-limiting examples of methods for measuring nicotine/alkaloid contentinclude such methods as gas chromatography, mass spectrometry (Domino etal. 1992 Med Sci Res. 20:859-860; Sheen et al. 2006 J Food Sci53(5):1572-1573), HPLC (Keinänen et al. 2001 J Agric Food Chem49:3553-3558; Halitschke and Baldwin 2003 Plant J 36: 794-807), UVabsorption (Willits et al. 2005 Analytical Chemistry 22:430-433) and thelike.

As used herein, the term “modulate,” “modulates,” modulated” or“modulation” refers to enhancement (e.g., an increase) or inhibition(e.g., a reduction) in the specified activity (e.g., modulated nicotineproduction/content).

As used herein, the terms “increase,” “increasing,” “increased,”“enhance,” “enhanced,” “enhancing,” and “enhancement” (and grammaticalvariations thereof), as used herein, describe an increase in thenicotine content of a plant as a result of the introduction into theplant of an isolated nucleic acid molecule or nucleic acid construct ofthe invention, thereby producing a transgenic plant having increasednicotine content. This increase in nicotine content can be observed bycomparing the nicotine content of the plant transformed with theisolated nucleic acid molecule or nucleic acid construct of theinvention to the nicotine content of a plant lacking (i.e., nottransformed with) the said nucleic acid molecule or nucleic acidconstruct of the invention and grown under the same environmentalconditions (i.e., a control). The increase can be measured as anincrease in percent by weight or as a percent increase of the controlvalue. Thus, in some embodiments, the nicotine content of a planttransformed with an isolated nucleic acid molecule or nucleic acidconstruct of the invention can be increased by at least about 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 100%, 125% 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%,500% or more as compared with a control. In some embodiments, thenicotine content of a plant transformed with an isolated nucleic acidmolecule or nucleic acid construct of the invention can be increased byat least about 30% as compared with a control. In other embodiments, thenicotine content of a plant transformed with an isolated nucleic acidmolecule or nucleic acid construct of the invention can be increased byat least about 50% as compared with a control. In still otherembodiments, the nicotine content of a plant transformed with anisolated nucleic acid molecule or nucleic acid construct of theinvention can be increased by at least about 100% as compared with acontrol.

In some embodiments, the nicotine content of a plant transformed with anisolated nucleic acid molecule or nucleic acid construct of theinvention can be about 10 mg/g (e.g., about 1%) to about 100 mg/g (e.g.,10%) (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mg/g) dry weight nicotine. In particular embodiments, the nicotinecontent of a plant transformed with an isolated nucleic acid molecule ornucleic acid construct of the invention can be about 20 mg/g (e.g.,about 2%) to about 100 mg/g (e.g., 10%) dry weight nicotine; about 30mg/g to about 100 mg/g dry weight nicotine, about 40 mg/g to about 100mg/g dry weight nicotine, about 50 mg/g to about 100 mg/g dry weightnicotine, and the like. In still other embodiments, the nicotine contentof a plant transformed with an isolated nucleic acid molecule or nucleicacid construct of the invention can be at least about 30 mg/g dry weightnicotine, at least about 40 mg/g (e.g., about 4%) dry weight nicotine,at least about 50 mg/g (e.g., about 5%) dry weight nicotine, at leastabout 60 mg/g (e.g., about 6%) dry weight nicotine, and the like.

As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,”“diminish,” “suppress,” and “decrease” (and grammatical variationsthereof), describe, for example, a decrease in the nicotine content of aplant of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 100%, or any range therein, as compared with a control asdescribed herein. Thus, in some embodiments, the nicotine content of aplant transformed with an isolated nucleic acid molecule or nucleic acidconstruct of the invention can be reduced by at least about 1%, at leastabout 10%, at least about 25%, at least about 50%, at least about 75%,at least about 95% and the like, as compared. In other embodiments, thenicotine content of a plant transformed with an isolated nucleic acidmolecule or nucleic acid construct of the invention can be reduced suchthat the control plant has a level at least about 2 fold to about 10fold (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 fold and the like)as compared to the transformed plant. In representative embodiments, thereduction is by about 5 fold to about 7 fold.

A further aspect of the invention provides transformed non-human hostcells and transformed non-human organisms comprising the transformednon-human cells, wherein the transformed cells and transformed organismscomprise nucleic acid molecules comprising one or more nucleotidesequences of the invention. In some embodiments, the transformednon-human host cell includes but is not limited to a transformedbacterial cell, and/or a transformed plant cell. Thus, in someembodiments, the transformed non-human organism comprising thetransformed non-human host cell includes, but is not limited to, atransformed bacterium, and/or a transformed plant.

In some particular embodiments, the invention provides a transgenicplant cell comprising a nucleic acid molecule of the invention and/or atransgenic plant regenerated from said transgenic plant cell.Accordingly, in some embodiments of the invention, a transgenic planthaving modulated (e.g., increased or reduce) nicotine content isprovided, said transgenic plant regenerated from a transgenic plant cellcomprising at least one isolated nucleic acid molecule/nucleic acidconstruct of the invention.

Additional aspects of the invention include a harvested product producedfrom the transgenic plants and/or parts thereof of the invention, aswell as a processed product produced from said harvested product. Aharvested product can be a whole plant or any plant part, as describedherein, wherein said harvested product comprises a recombinant nucleicacid molecule/construct of the invention. Thus, in some embodiments,non-limiting examples of a harvested product include a seed, a fruit, aflower or part thereof (e.g., an anther, a stigma, and the like), aleaf, a stem, and the like. In other embodiments, a processed productincludes, but is not limited to, cigarette, cigarette tobacco, cigartobacco, a cigar, pipe tobacco, chewing tobacco, leaf tobacco, shreddedtobacco and cut tobacco, and the like produced from a transgenic plantof the invention.

A plant useful with this invention can be any plant that producesnicotine and/or other related alkaloids. Thus, in some embodiments, theplant can be Nicotiana tabacum, Nicotiana rustica or Nicotianabenthamiana. Any variety of tobacco is useful with this inventionincluding, but not limited to, Aromatic Fire-cured, Brightleaf tobacco,Burley; Cavendish; Corojo; Criollo; Oriental Tobacco; Perique; Shadetobacco; Thuoc lao; Type 22; NC95, K326, K346, White Burley, WildTobacco, Y1, and the like.

Thus, in some particular embodiments, a transgenic plant of theinvention includes, but is not limited to, a transgenic tobacco plant orpart thereof is provided, wherein the nicotine content of said plant orpart thereof is modulated.

As used herein, the term “plant part” includes but is not limited toembryos, pollen, ovules, seeds, leaves, flowers, branches, fruit,kernels, ears, cobs, husks, stalks, roots, root tips, anthers, plantcells including plant cells that are intact in plants and/or parts ofplants, plant protoplasts, plant tissues, plant cell tissue cultures,plant calli, plant clumps, and the like. Further, as used herein, “plantcell” refers to a structural and physiological unit of the plant, whichcomprises a cell wall and also may refer to a protoplast. A plant cellof the invention can be in the form of an isolated single cell or can bea cultured cell or can be a part of a higher-organized unit such as, forexample, a plant tissue or a plant organ. A “protoplast” is an isolatedplant cell without a cell wall or with only parts of the cell wall.Thus, in some embodiments of the invention, a transgenic cell comprisinga nucleic acid molecule and/or nucleotide sequence of the invention is acell of any plant or plant part including, but not limited to, a rootcell, a leaf cell, a tissue culture cell, a seed cell, a flower cell, afruit cell, an embryo cell, an ovule cell, a pollen cell, and the like.

In some particular embodiments, the invention provides a transgenic seedproduced from a transgenic plant of the invention, wherein thetransgenic seed comprises a nucleic acid molecule/nucleotide sequence ofthe invention.

“Plant cell culture” means cultures of plant units such as, for example,protoplasts, cell culture cells, cells in plant tissues, pollen, pollentubes, ovules, embryo sacs, zygotes and embryos at various stages ofdevelopment. In some embodiments of the invention, a transgenic tissueculture or transgenic plant cell culture is provided, wherein thetransgenic tissue or cell culture comprises a nucleic acidmolecule/nucleotide sequence of the invention.

As used herein, a “plant organ” is a distinct and visibly structured anddifferentiated part of a plant such as a root, stem, leaf, flower bud,or embryo.

“Plant tissue” as used herein means a group of plant cells organizedinto a structural and functional unit. Any tissue of a plant in plantaor in culture is included. This term includes, but is not limited to,whole plants, plant organs, plant seeds, tissue culture and any groupsof plant cells organized into structural and/or functional units. Theuse of this term in conjunction with, or in the absence of, any specifictype of plant tissue as listed above or otherwise embraced by thisdefinition is not intended to be exclusive of any other type of planttissue.

The invention further provides a plant crop comprising a plurality oftransgenic plants of the invention planted together in an agriculturalfield. In some embodiments, the plant crop comprises a plurality oftransgenic tobacco plants of the invention planted together in anagricultural field.

“Genetic engineering” encompasses any methodology for introducing anucleic acid or specific mutation into a host organism. For example, aplant is genetically engineered when it is transformed with apolynucleotide sequence that suppresses expression of a gene, such thatexpression of a target gene is reduced compared to a control plant. Aplant is genetically engineered when a polynucleotide sequence isintroduced that results in the expression of a novel gene in the plant,or an increase in the level of a gene product that is naturally found inthe plants. In the present context, “genetically engineered” includestransgenic plants and plant cells, as well as plants and plant cellsproduced by means of targeted mutagenesis effected, for example, throughthe use of chimeric RNA/DNA oligonucleotides, as described by Beetham etal., Proc. Natl. Acad. Sci. U.S.A. 96: 8774-8778 (1999) and Zhu et al.,Proc. Natl. Acad. Sci. U.S.A. 96: 8768-8773 (1999), or so-called“recombinagenic oligonucleobases,” as described in International patentpublication WO 2003/013226. Likewise, a genetically engineered plant orplant cell may be produced by the introduction of a modified virus,which, in turn, causes a genetic modification in the host, with resultssimilar to those produced in a transgenic plant, as described herein.See, e.g., U.S. Pat. No. 4,407,956. Additionally, a geneticallyengineered plant or plant cell may be the product of any native approach(i.e., involving no foreign nucleotide sequences), implemented byintroducing only nucleic acid sequences derived from the host plantspecies or from a sexually compatible plant species. See, e.g., U.S.published patent application No. 2004/0107455.

“Tobacco product” refers to a product comprising material produced by aNicotiana plant, including for example, nicotine gum and patches forsmoking cessation, cigarette tobacco including expanded (puffed) andreconstituted tobacco, cigar tobacco, pipe tobacco, cigarettes, cigars,and all forms of smokeless tobacco such as chewing tobacco, snuff, snusand lozenges. “Cigarettes” includes electronic cigarettes and “heat notburn” products which are cigarette-like devices that heat tobacco ratherthan burn tobacco.

The invention will now be described with reference to the followingexamples. It should be appreciated that these examples are not intendedto limit the scope of the claims to the invention, but are ratherintended to be exemplary of certain embodiments. Any variations in theexemplified methods that occur to the skilled artisan are intended tofall within the scope of the invention.

EXAMPLES Example 1. Materials and Methods

A. Yeast One-Hybrid Experiments for Cloning Transcription Factors

Bait Vector Construction

A yeast one-hybrid system, Matchmaker™ One-Hybrid Library Constructionand Screening Kit (Clontech, Mountain View, Calif.), was employed toscreen for transcription factors that bind to the promoter of thetobacco QPT2 gene (U.S. Pat. No. 5,837,876). The promoter, 1034 bp inlength, was cleaved from pTobRD2-PMT_(OX) construct provided by the22^(nd) Century LLC. (Buffalo, N.Y.), and inserted upstream of the GAL4minimal promoter in vector pHIS2.1 to form the bait construct pTobHis.The insert was first verified by BamHI digestion and the orientation ofthe promoter was confirmed by PCR using the following primers designedfrom sequence of the QPT2 promoter and His3 gene:

Tob1F: (SEQ ID NO: 9) 5′-ACATCTGTAACCGGAACAGCAC-3′ His1R:(SEQ ID NO: 10) 5′-GGTCGTCTATGTGTAAGTCACC-3′

Yeast strain Y187 was transformed with the bait construct pTobHis and100 μL of 1/15 dilution of the original transformants was plated onSD/-Trp and SD/-Trp/-His media, respectively. The plates were titratedwith a series of 3-AT (3-amino-1,2,4-triazole) (0, 5, 10, 15, 20, 30,50, 75, 100 mM) to optimize the concentration that suppresses the basalexpression of the bait construct. The plates were cultured at 30° C. forfive days.

cDNA Library Construction

Total RNA was extracted from roots of two-month-old greenhouse-growntobacco plants (cv. NC95) 0.5 hr after topping. An oligo(dT) primer,modified by fusion with a 25-mer sequence, called CDS III, and theSMART™ III primer from the kit were used for synthesis of the firststrand cDNA so to flank the synthesized cDNA with the two primersequences. Long distance PCR (LD-PCR) was performed to amplify thesynthesized cDNA using the Advantage 2 PCR kit (Clontech) with the CDSIII and SMART™ III primers based on the manufacturer's instruction. ThePCR product was examined on agarose gel and the double-stranded cDNA waspurified with CHROMA SPIN TE-400 columns from the kit and concentrated.

Yeast Transformation and Positive Colony Selection

Yeast competent cells were prepared and transformed with the bait vectorpTobHis, the linearized prey vector pGADT7-Rec2, and the ds cDNA (˜4 μg)following the manufacturer's instructions. The homologous recombinationbetween the prey vector and the ds cDNA took place at the CDS III andSMART™ III sites of the linearized prey vector inside the yeast cells.The transformed cells were propagated, collected, and spread on DDOmedium (SD/-Leu/-Trp) to estimate the screening efficiency of theco-transformation, and on TDO medium (SD/-Leu/-Trp/-His) to identifycolonies positive in the one-hybrid selection.

Characterization of Positive Colonies from Yeast One-Hybrid Selection

The insert length of the positive colonies was evaluated by a standard30-cycle PCR (Bioneer, Alameda, Calif.) with 1 μL overnight yeastculture and the following two primers provided by the kit:

5′ PCR Primer: (SEQ ID NO: 11) 5′-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGG-3′3′ PCR Primer: (SEQ ID NO: 12)5′-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGGCCGAC-3′

The PCR products were examined on agarose gels, and all the preyconstructs with cDNA inserts longer than 500 bp were subjected toplasmid isolation with QIAprep Spin Miniprep kit (Qiagen, Valencia,Calif.). The plasmids isolated from yeast colonies were subsequentlytransformed into E. coli DH5α for propagation. The prey plasmids wereisolated again from E. coli and the cDNA inserts were subjected tosequence analysis using the T7 primer and the 3′PCR primer. Any yeastcolony which contained more than one prey constructs was re-streaked onSD/-Leu for 2-3 times until the PCR result yields a single amplifiedfragment.

The sequences of the cDNA inserts were used for BLAST analysis with theNCBI GenBank database. A total of five transcription factors wereidentified from about 100 positive colonies. However, none of these fivetranscription factor cDNAs was in full length but they all had poly(A)tails.

Recovery of Full-Length cDNAs of the TFs

RNA was isolated from root tissue of greenhouse grown NC95 tobaccoplants 0.5 hr after topping. The GENERACER™ kit (Invitrogen, Carlsbad,Calif.) was used to obtain the missing 5′ sequences of the TF cDNAsfollowing the manufacturer's manual. One or two gene specific primerswere designed based on the sequences near the 5′ termini of the partialcDNAs of the six isolated TF genes for PCR together with the GENERACER™5′primer provided by the kit. The sequences of these gene specificprimers are:

NtERF GSP1: (SEQ ID NO: 13) 5′-CTATCTCCGACTTCTGGTCTTCCTCT-3′ NtERF GSP2:(SEQ ID NO: 14) 5′-CCACGGTCTCTGCCTTATTCCTCTGTA-3′ NtMYC2a GSP1:(SEQ ID NO: 15) 5′-ACACATTTGGTACAACAGCTCTAAGTGC-3 NtMYC2a GSP2:(SEQ ID NO: 16) 5′-TGCAATTGCATCACCAAGAAGTGATGCT-3′ NtMYC2b GSP:(SEQ ID NO: 17) 5′-CGGGGAGTTGGTGTAGTAG-3′ NtARF GSP1: (SEQ ID NO: 18)5′-CCTTTTGTGTCTCCCTTCCTACTGATG-3′ NtARF GSP2: (SEQ ID NO: 19)5′-CTAAGTTTTGAGAGCACTGGGTCCCAAG-3′

After the full-length cDNAs were recovered, primers were designed toobtain the full-length coding sequences of these four transcriptionfactors by PCR:

NtERFFL1F: (SEQ ID NO: 20) 5′-TCTAGAGGATCCCGGGATGTGTGGAGGTGCCATAATCC-3′NtERFFL1R: (SEQ ID NO: 21) 5′-GCGCCCGGGTTCAGTAAAACAGCTGCTGCTGC-3′NtARFFL1F: (SEQ ID NO: 22) 5′-GGATCCATGATGTGTGGACTTATTGATC-3′ NtARFFL1R:(SEQ ID NO: 23) 5′-CCCGGGCTACAAAGCAATATCAAGAATC-3′ NtMYC2a 1F:(SEQ ID NO: 24) 5′-GCGGTCTAGACAGATCTGAATTGATTTGTCT-3′ NtMYC2a 1R:(SEQ ID NO: 25) 5′-GCGGTCTAGAACATTATTCAGAGCTCACTATG-3′ NtMYC2b 1F:(SEQ ID NO: 26) 5′-GCGTCTAGAATGACGGACTATAGAATACCA-3′ NtMYC2b 1R:(SEQ ID NO: 27) 5′-GCGTCTAGATCATCGCGATTCAGCAATTCT-3′

All PCR reactions were performed using the high-fidelity Taq DNApolymerase (Phusion, Finnzymes, Espoo, Finland). The PCR products werecloned into pCRBluntII-TOPO or pCR4-TOPO vector (Invitrogen) forsequence analysis by a commercial provider.

B. Expression Analysis of the Isolated Transcription Factors in Tobacco

Tobacco plants (cv. NC 95), grown in the greenhouse for about two monthsuntil just before flowering, were subjected to gene expression analysisin various organs as well as in roots after topping, wounding, or MeJA(Sigma, St. Louis, Mo.) treatments. Topping was performed by cutting offthe shoot apex of plants immediately below the bud. Wounding wasperformed by cutting leaves on the plants with scissors: five cuts perleaf on top three fully-grown leaves. For the MeJA treatment, 50 μMsolution (about 5 mL) was sprayed on all the plant leaves.

Root tissues were collected at the time points of 0 h, 0.25 h, 0.5 h, 1h, 2 h, 4 h, and 6 h after treatments. RNA samples were isolated andNorthern blot analysis was performed as described in Chapter 1. Theprimers designed from the coding sequences used to generate the probesfor Northern hybridization were:

NtERFNOR F: (SEQ ID NO: 28) 5′-ACACTGCACTAGCACCATCCC-3′ NtERFNOR R:(SEQ ID NO: 29) 5′-CTGCATTGTACTACGTACTACC-3′ NtMYC2a/bNOR F:(SEQ ID NO: 30) 5′-GAAGTAACGGATACTGAATGG-3′ NtMYC2a/bNOR R:(SEQ ID NO: 31) 5′-ATCCTTGTGTTTGCTGAGAAT-3′ NtARFNOR F: (SEQ ID NO: 32)5′-CTGCCTATAGCCAACTGTTG-3′ NtARFNOR R: (SEQ ID NO: 33)5′-AAGCTGCTGGATACAGGAGC-3′C. Tobacco Transformation for Over-Expression and Down-Regulation of theIsolated Transcription Factors

pBI121 was used as the backbone vector to make the over-expression andRNAi gene constructs. The coding sequences of the isolated transcriptionfactors were cleaved from their cloning vectors (pCRBluntII-TOPO orpCR4-TOPO) and inserted into pBI121 at the place of the GUS gene to beunder control of the constitutive CaMV 35S promoter (FIG. 1).

The RNAi technique was used to down-regulate the individual TFs intransgenic tobacco plants. For RNAi vector construction, the partialcoding sequence of each TF was obtained by PCR with the following pairsof the primers:

NtERFRNAiF: (SEQ ID NO: 34) 5′-GACTGAGCTCTCTAGAGTGGAGGTGCCATAATCCCCGA-3′NtERFRNAiR: (SEQ ID NO: 35)5′-GACTCCCGGGGATATCCGGTCTCTGCCTTATTCCTCTGTA-3′ NtMYC2RNAiF:(SEQ ID NO: 36) 5′-GGGGAGCTCTCTAGAGCTGCAACAGCGACTCCAGA-3′ NtMYC2RNAiR:(SEQ ID NO: 37) 5′-ATT CCCGGGGTCGACCCGTTAACAAACGATTGAGTC-3′ NtARFRNAiF:(SEQ ID NO: 38) 5′-GGGGAGCTCGGATCCGATGGGATTGCAGTATCAGAC-3′ NtARFRNAiR:(SEQ ID NO: 39) 5′-GGGACTAGTGTCGACGAGTACTTGGATTGCAATGAC-3′

The PCR fragment was first cloned into pQLi to replace the red and blueregions (as shown in FIG. 2) to create inverted repeat, and then thewhole cassette was inserted into pBI121 to replace the GUS gene. All theresultant over-expression and RNAi vectors were confirmed by appropriaterestriction digestions and sequencing analysis. FIG. 2 provides aschematic representation of the region of vector pQLi used for invertedrepeat cloning.

Agrobacterium-mediated tobacco transformation was performed usingAgrobacterium strain LBA4404 and a leaf disc protocol (Horsch et al.(1985) Science 227: 1229-1231). Small leaf pieces from the plants to betransformed were mixed with overnight-grown Agrobacterium tumefaciensculture (OD₆₀₀=1.0) for 2 min. Excess Agrobacterium culture solution wasremoved using sterile paper towel. Co-cultivation was conducted for 2-3days on MS medium. MS medium with BA (1 mg/L), IBA (0.1 mg/L) andkanamycin (100 mg/L) was used as the selection medium. Subculturing wasperformed 2-3 times at 2-wk interval. Non-transformed control plantswere also regenerated using the same media and procedure but withoutantibiotic selection. The rooting medium was MS with 50 mg/L kanamycinand no hormones. Putative transgenic tobacco plants (cv. NC 95) weregrown in the greenhouse for about two months and root tissues werecollected just before flowering.

Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad,Calif.) according to the manufacturer's protocol. Approximately 700 mgroot tissue or 200 mg leaf tissue were used for RNA isolation. ForNorthern blot analysis, the extracted RNA was dissolved in DEPC-treatedwater and quantified with the Nanodrop (ND-1000). Ten μg RNA wasseparated on a 1% agarose gel in MOPS buffer. The gel was stained withEtBr and the image was photographed under UV illumination. Separated RNAon the gel was blotted onto the Hybond-N+ nylon membrane. The primersused to generate probes were the same as described above.

D. qRT-PCR Analysis of Pathway Gene Expression

Root was collected from T₀ transgenic plants before flowering. RNA wasisolated from root tissue using TRIzol reagent (Invitrogen) according tothe manufacturer's manual and first strand cDNA was synthesized usingSuperScript™ III ReverseTranscriptase (Invitrogen) with an oligo(dT)primer. qRT-PCR was performed using FastStart Universal SYBR GreenMaster (Rox) (Roche, Mannheim, Germany) on ABI7900 (Applied Biosystems,Foster City, Calif.). Tobacco actin gene was chosen as a control fornormalization. Three technical replicates were performed with RNAsamples from each transformation event using the following PCR program:50° C. for 1 min; 95° C. for 15 sec; 40 cycles of 95° C., 15 secfollowed by 63° C., 1 min with the primers for pathway genes listed inTable S3. Two transformation events per gene construct were analyzed.The means and standard errors are presented.

E. Electrophoretic Mobility Shift Assays (EMSA)

Since the yield of the full length MYC2a protein when expressed in E.coli was very low, we used the N-truncated MYC2a protein for EMSA.N-truncated MYC2a protein (MYC2aΔN, from No. 264 to No. 659 AA) wasexpressed in fusion with GST in Rosetta 2(DE3) Singles (Novagen,Madison, Wis.). The cell culture was treated with 0.2 mM IPTG for 4 h at15° C. to induce protein expression.

The recombinant protein was purified using glutathione-agarose beads(Sigma) and the MYC2aΔN was cleaved and eluted from the beads withthrombin (Sigma). Thrombin was removed from MYC2aΔN solution withpAminobenzamidine-Agarose (Sigma) and the MYC2aΔN was concentrated byAmicon Ultra centrifugal filter units Ultra-15 (MWCO 10 kDa) (Millipore,Billerica, Mass.).

Probe was labeled according to the manual of kit (Biotin 3′ End DNALabeling Kit, Pierce, Rockford, Ill.). Two complementary oligomers werelabeled on the 3′ends. Double strand DNA was prepared by mixing of equalamount of labeled complementary oligomers. Mixture was heated at 95° C.for 5 min and cooled down to room temperature for 1 h.

G2-box probe: (SEQ ID NO: 40)-189 5′-AGTAGCTGAACACGTTTTATTTATGGTTGTTGAATA GT-3′-227 (SEQ ID NO: 41)3′-TCATCGACTTGTGCAAAATAAATACCAACAACTTATCA-5′ Mutated G2-box cold probe:(SEQ ID NO: 42) 5′-AGTAGCTGAATCACATTTATTTATGGTTGTTGAATAGT-3′(SEQ ID NO: 43) 3′-TCATCGACTTAGTGTAAATAAATACCAACAACTTATCA-5

EMSA was performed according to manual of the kit (LightShift®Chemiluminescent EMSA Kit, Pierce). The binding buffer was 10 mM Tris,50 mM KCl, 1 mM DTT, 0.05 μg/μl poly(di-dc), 0.05% NP-40, and 2.5% (v/v)glycerol. The probe was 20 fmol for each reaction. The cold probe andmutated probe were 10 pmol (500×) and the protein was 0.75 μg MYC2aΔN.The binding reaction was performed at room temperature for 20 min. Thereaction mixture was separated on a 5% polyacrylamide gel and blotted onHybond™-N⁺ membrane (Amersham, Piscataway, N.J., USA). UV crosslinkerwas used to crosslink the DNA to membrane. The membrane was probed andsignal was detected according to the manual of kit (ChemiluminescentNucleic Acid Detection Module Kit, Pierce). Kodak BioMax MS film(Carestream Health, Inc. Rochester, N.Y.) was exposed to record thesignal.

F. Quantification of Major Alkaloids

Nicotine levels in dried leaves of the transgenic plants were kindlyquantified by gas chromatography. Each sample was prepared by placing0.2000±0.0010 g of dried ground tobacco leaves into a 50 mL Erlenmeyerflask. Two mL of 2N NaOH solution was added to each flask and swirled tomoisten the tobacco. After 15 min of rest, 10 mL of methyl tertiarybutyl ether (MTBE) containing 0.1062 g/mL of quinoline was added to theflask. The flasks were placed on a shaker for 2.5 hrs. After shaking theflasks were allowed to sit overnight to separate. Approximately 1 mL ofthe top MTBE layer was transferred into a vial. GC analysis wasconducted using a split injection (40:1) on an Agilent HP 6890 GC-FID(Agilent Technologies, Santa Clara, Calif.) using a 30 meter DB-5MScolumn (0.53 mm ID and 1.5 μm film thickness). The carrier gas washelium at a linear velocity of approximately 38 cm/sec. The injector anddetector were both set at 250° C. The analysis consists of a temperatureprogram from 110° C. initially held for 0.5 min followed by a ramp to280° C. at a rate of 25° C./min where the final temperature was held for20 min. Data were collected and analyzed using Agilent Chemstationsoftware. A multi point internal standard calibration table wasconstructed for each compound. The curves for each compound are asfollows:Y=2.32779e-1*x+8.05332e-3  Nicotine:Y=2.26220e-1*x−3.49890e-3  Nornicotine:Y=2.23584e-1*x+2.27888e-4  Anabasine:Y=1.33963e-1*x−3.08881e-3  Anatabine:

Example 2. Identification of the Transcription Factors

The yeast one-hybrid technique was used in this study to identify thetranscription factors. This technique includes three importantcomponents: bait construct, prey vector, and cDNA. The QPT2 promoterregion was inserted upstream the GAL4 minimal promoter in the baitvector, which drives a histidine synthesis gene HIS3. Thus, if the yeastcell contains the bait vector and a prey vector expressing a TF-GAL4 AD(activation domain) fusion protein that binds to the QPT2 promoter, theHIS3 gene will express and the cell will be able to grow on a screeningmedium which lacks histidine. The bait construct was confirmed byrestriction digestion and PCR for the insertion of the QPT2 promoter andits orientation. It was then tested for leaking expression of HIS3 (orbasal expression because of endogenous yeast transcription factors).3-amino-1,2,4-triazole (3-AT) is a competitive inhibitor of the reportergene HIS3 product, a histidine biosynthetic enzyme. A titrationexperiment of 3-AT was conducted to optimize 3-AT concentration in theyeast culture medium to minimize potential false positives in thescreening experiments. No colony grew on the medium (SD/-His/-Trpcontaining a series of 3-AT from 5 mM to 75 mM) while many colonies grewon the control plate of SD/-Trp. This experiment was repeated and thesame result was obtained. Thus, it was not necessary to add 3-AT to thescreening medium to suppress the basal expression of HIS3 gene and thebackground growth.

Total RNA was extracted from the root tissue collected 30 min aftertopping and used to make a cDNA library for the yeast one-hybridscreening. Many of the nicotine biosynthetic pathway genes are inducedseveral hrs after topping and it was expected that the transcriptionfactor genes would be induced earlier. A time of 30 min after toppingwas estimated to be appropriate to capture a “snapshot” of theexpression of these transcription factors. The quality of the cDNA wasexamined by PCR.

A total of three screening experiments were performed by transformingyeast competent cells with the bait vector, the prey vector, and thecDNA collection (homologous recombination would take place between theprey vector and the cDNA inside the yeast cell so the cDNA-GAL4 ADfusion gene will express from the prey vector), and approximately 1.6million yeast colonies were screened. After seven days of incubation,actively-growing yeast colonies were selected for colony PCR to screenfor prey plasmid which has the cDNA insert longer than 500 base pair.All yeast colonies which contained more than one prey plasmid (more thanone amplified DNA fragment on agarose gels) were subjected to successivere-streaking on SD/-Leu medium until only one prey plasmid was left inthe colony. The isolated prey plasmid was used to transform E. coli(strain DH5α) for plasmid propagation. Approximately 100 yeast positivecolonies were finally isolated and sequenced, among which five wereputative transcription factors as identified based on the BLAST analysiswith the NCBI GenBank database. These five transcription factors wereshown to belong to five TF families: GRAS, AP2/ERF, bHLH, ARF and WRKY.

All the cloned TF cDNAs were partial in length with their 5′ sequencesmissing. To obtain full-length cDNAs for these TFs, the 5′ RACE (RapidAmplification of cDNA Ends, Invitrogen, Carlsbad, Calif.) technique wasperformed, and high-fidelity Taq DNA polymerase was used in all the PCRreactions. For each TF, at least five randomly picked colonies weresubjected to sequence analysis. During the process, another bHLH genewith high homology to the cloned one was identified and cloned. Thesequences and expression patterns of these TF genes were characterized.To test whether these transcription factors have effects on nicotinebiosynthesis pathway, transgenic over-expression and RNAi lines of allthe six transcription factors were produced. Transformed plants weregrown in the greenhouse for about two months. Total RNA was extractedfrom root tissue and subjected to Northern blot analysis of pathway geneexpression and nicotine concentration.

Example 3. Transcription Factor NtMYC2a and NtMYC2b Genes

Two full-length bHLH transcription factor cDNAs were cloned with 2214and 2391 bp in length and encoding 659 and 658 AA, respectively (see,Appendix). They share 96% identity at the cDNA sequence level. The twoTFs were named NtMYC2a and NtMYC2b.

A. NtMYC2 Expression Pattern in Tobacco

Northern blot analysis (FIG. 3) shows NtMYC2 genes expressed in tobaccoroot, stem, leaf and flower. Total RNA isolated from various organs offully grown plant was probed with NtMYC2b PCR fragment probes. The 25SrRNA stained with EtBr in gel is also shown as a loading reference. Dueto the high homology between the two MYC2 genes, the probe for all theNorthern blot analysis of MYC2 is a partial coding sequence of 505 bpfrom MYC2b, and cannot distinguish the two genes and thus they wereanalyzed together. Northern blot analysis shows that, despite nicotinebiosynthesis occurring only in root, and the pathway genes, such asNtPMT, NtQPT2, and NtMPO, expressing only in root, NtMYC2 has expressionin all the four organs (root, stem, leaf and flower) examined withoutany treatment, and the highest expression in stem (FIG. 3). NtMYC2expression in all major organs indicates the TFs also function inmetabolic pathways other than nicotine biosynthesis (Dombrecht et al.(2007) Plant Cell 19: 2225-2245).

B. NtMYC2 Expression Patterns in Tobacco Root After Topping, Wounding,and MeJA Treatment

Since nicotine accumulation was induced by topping, wounding, or MeJAtreatment, expression of the NtMYC2 genes in root after these individualtreatments was investigated. Two-month-old tobacco plants (just beforeflowering) were treated with topping, wounding or MeJA, and the roottotal RNAs were used for northern analysis. Northern hybridization showsa time course induction pattern of NtMYC2 after each treatment. Theprobe is from NtMYC2b. The 25S rRNA stained in gel with EtBr is shown asa loading reference. As shown in FIG. 4, NtMYC2 expression was inducedin root by topping, wounding, or MeJA treatment. Compared to thecontrol, the induced expression of NtMYC2 seems to be biphasic: theexpression increased 0.25 h after the treatments, declined slightlyafterwards, and increased again at 4 or 6 h time point, with woundinghaving the strongest induction effect among the three treatments.

C. NtMYC2a Binds to the G2 Box in NtQPT2 Promoter

Three G-boxes (G2, G3 and G4) were identified in the 0.6 kb NtQPT2promoter (NCBI accession No. AJ748263), among which G2 box has thehighest binding strength with NtMYC2b (Shoji and Hashimoto, 2011). Tovalidate that NtMYC2a binds to G2-box, EMSA experiment was performedwith probes of 38 bp consisting of a G2 box (CACGTT) and flankingregions from the NtQPT2 promoter and mutated probe with mutation in theG2 box sequence. We cloned the full length of NtMYC2a and expressed therecombinant protein (NtMYC2a fused with GST) in E. coli. Due to the verylow yield of the full length NtMYC2a protein, we truncated it and onlyexpressed the C-terminal bHLH domain of the NtMYC2a protein (NtMYC2aΔN,from No. 264 to No. 659 AA).

The binding experiment showed that NtMYC2aΔN specifically binds to theG2 box in the NtQPT2 promoter in vitro (FIG. 5). Dilution with coldprobe reduced the binding signals whereas dilution with mutated coldprobe maintained most of the signals.

D. Analysis of NTMYC2a and NtMYC2b Transgenic Lines

Seven NtMYC2a and nine NtMYC2b over-expression lines and nine NtMYC2RNAi transgenic lines, in which both genes are expected to besuppressed, were generated to test the effect of these two TF genes onnicotine biosynthesis. The 336 bp fragment used for RNAi construct wasfrom NtMYC2b coding sequence, which shares 94% identity with that regionof NtMYC2a gene.

FIG. 6 shows the expression levels of NtMYC2a and NtMYC2b in transgenicplants over-expressing NtMYC2a or NtMYC2b gene. Compared to the wildtype and vector control, two lines of NtMYC2a (AOE-3, and 6) and sevenlines of NtMYC2b (BOE-7, 10, 11, 13, 14, 16, and 17) were clearlyover-expression lines.

FIG. 7 shows the effects of the RNAi construct on NtMYC2a/b expression.Expression level of NtMYC2 in three RNAi lines (RNAi-1, 2, 3) wasgreatly reduced. All other lines did not show substantial changes inNtMYC2 expression.

Based on the Northern analysis, nicotine levels of two NtMYC2aover-expression and six NtMYC2b over-expression lines with hightransgene expression and three RNAi lines with much lower expression ofNtMYC2 were quantified (FIG. 8).

FIG. 8 shows that three NtMYC2b over-expression lines (BOE-10, 16, and17) and two NtMYC2a over-expression lines (AOE-3 and AOE-6) had highernicotine level than the controls (41% to 149% higher than vectorcontrol). However, other lines with higher expression level of NtMYC2b,such as BOE-11, 13, and 14 didn't have higher nicotine level. The threeRNAi lines (RNAi-1, 2 and 3) showed much lower nicotine level (aboutfive-fold less) than the controls.

Because over-expression and knock down of the NtMYC2 genes altered boththe nicotine and total alkaloid levels (data not shown), selectedover-expression and RNAi lines were subjected to Northern blothybridization analysis and qRT-PCR analysis to evaluate the effects onnicotine biosynthesis pathway genes' expression. FIGS. 9, 10 and 11 showthe expression of QPT and PMT genes as affected by the two transcriptionfactors.

Over-expression of NtMYC2b did not alter the mRNA levels of the pathwaygenes with the exception of moderate expression reduction of the NtPMT,NtA622 and NtNBB1 genes. However, the expression of all pathway geneswas reduced by over-expression of NtMYC2a. RNAi silencing of both NtMYC2genes resulted in decrease of the expression levels of all pathway genesexcept NtADC.

Surprisingly, the steady-state mRNA levels of all seven pathway genes inNtMYC2a over-expression lines (AOE-3 and AOE-6) were lower than control,with NtQPT, NtMPO and NtADC even lower than in the RNAi lines. Incontrast, changes at mRNA levels of these genes in NtMYC2bover-expression lines were not obvious except for NtA622 and NtNBB1,expressions of which were about 60% of the control's (FIG. 5).Interestingly, among these seven genes investigated, NtADC expressionlevels were only reduced in AOE lines and virtually not affected in RNAiand BOE lines. Considering it is not a nicotine biosynthesis-dedicatedgene, it is sensible that its expression is also regulated by anothertranscription circuit.

From the data on over-expression and RNAi lines, it is evident thatNtMYC2a and/or NtMYC2b genes modulate nicotine biosynthetic pathway geneexpression and nicotine level. It is more remarkable that in the threeRNAi lines (RNAi-1, -2, and -3) which had NtMYC2 expressionsubstantially decreased, both PMT and QPT mRNA levels were reduced byapproximately ten-fold, and the nicotine level decreased aboutfive-fold. More interestingly, although NtMYC2a and NtMYC2b are highlyhomologous, their functions appear to be diverse: Over-expression ofNtMYC2a led to greatly reduced PMT and QPT mRNA levels yet the highestnicotine level (more than two-fold of the controls' and around 1% leafdry weight) whereas over-expression of NtMYC2b caused little change inPMT and QPT mRNA levels among the four lines analyzed while nicotineconcentration had a moderate increase (nearly 50%) in three out of thefour lines. Moreover, it is surprising to see that plants having thehighest nicotine level (AOE-3 and -6) were the ones with the lowest PMTand QPT mRNA levels, and plants with the lowest level of nicotine(RNAi-1, -2, and -3) had slightly more nicotine (but much reduced incomparison to the controls), and PMT and QPT mRNA. The data suggest thatthe nicotine level may not necessarily be associated with PMT and QPTmRNA levels.

E. Inheritance of Nicotine Content in T₁ NtMYC2a and NtMYC2b TransgenicLines

To further evaluate the functions of both NtMYC2 genes and the relatedtrait inheritance, T₁ plants from lines of AOE-3, AOE-6, BOE-16, BOE-17,RNAi-1, RNAi-2, and vector control were grown in greenhouse for abouttwo months and PCR was used to screen out the non-transgenic segregates.Leaf nicotine level was measured in non-topped plants and plants 10 daysafter topping separately. The results showed that non-topped transgenicNtMYC2aOE (AOE-3 and AOE-6) plants, in average, had 2.4 folds ofnicotine level of the vector control while NtMYC2bOE lines (BOE-16 andBOE-17) had 1.4 folds of nicotine content. RNAi (RNAi-1 and RNAi-2)plants contained only 14% nicotine content of the vector control plants.Similar increases were observed in the topping treatment. These results(FIG. 12) are highly consistent with those of T₀ plants (FIG. 8),indicating that the gained trait was inherited.

In both treatments, nicotine level in NtMYC2aOE plants was shown to besignificantly higher than that in NtMYC2bOE plants (p<0.0001),indicating these two closely-related paralogs function differentially innicotine biosynthesis.

F. Contents of Other Alkaloids in T1 NtMYC2a and NtMYC2b Over-ExpressionLines

The contents of nornicotine, anabasine and anatabine were also tested inT1 plants of AOE-3, AOE-6, BOE-16, BOE-17 and vector control lines (see,FIG. 13).

As shown in FIG. 13, with topping, the levels of each of these alkaloidsincreased significantly in both NtMYC2a and NtMYC2b over-expressionlines compared to vector control. When the plants were not topped, onlyanatabine had significant increase in NtMYC2a and NtMYC2bover-expression lines. Nornicotine and anabasine were significantlyincreased only in NtMYC2a but not NtMYC2b over-expression lines.Overall, the contents of all three alkaloids in NtMYC2a OE plants weresignificantly higher than those in NtMYC2b OE lines with bothnon-topping and topping treatments.

Example 4. Transcription Factor NtERF98

The full-length ERF transcription factor cDNA was 1019 bp in length andencodes a protein of 257 AA. The role this gene plays in the nicotinebiosynthesis has not been reported.

A. Expression Pattern of NtERF98 in Different Organs of the Plant

The expression pattern of NtERF98 in root, stem, leaf, and flower inwild type NC95 tobacco plants were examined. FIG. 14 shows that NtERF98was mainly expressed in the root, stem, and flower, and had littleexpression, if any, in leaves.

B. Expression Pattern of NtERF98 Under Treatments of Topping, Wounding,and MeJA

Since nicotine accumulation is induced by topping, wounding, and JAtreatment, these three treatments were applied individually in thisstudy to test whether NtERF98 expression was also affected by thesetreatments. Root tissues were collected 0.25 h, 0.5 h, 1 h, 2 h, 4 h and6 h after treatments. FIG. 15 shows the gene expression pattern in rootsof the treated tobacco plants.

NtERF98 expression under these three treatments had a similar biphasicdecline pattern. The steady-state NtERF98 mRNA level was reduced within15 min after initiation of each of these treatments. From 0.25 h to 0.5h, the expression level increased up to the basal level of the wild typecontrol in all three treatments. After 0.5 h, the mRNA levels were indecline again in the topping and wounding treatments. In MeJA treatment,the expression level reached its peak at 1 h, and declined thereafter.

C. Analysis of Transgenic Overexpression Lines

FIG. 16 shows the Northern analysis of NtERF98 and QPT in NtERF98putative over-expression lines. Six out of 10 lines showed much higherexpression level of NtERF98. Among the six NtERF98 over-expressionlines, five had reduced QPT expression. OE-19 and OE-25 showed similarpattern of expression of these two genes as the control plant, which hadvery low expression of NtERF98 and high expression of QPT. A negativecorrelation seems to exist between the expression pattern of NtERF98 andQPT in most of the transgenic plants that over-expressed NtERF98.

Leaf nicotine concentration of all the transformed lines weredetermined. FIG. 17 shows that the nicotine levels in the transformedover-expression lines of NtERF98 increased except for the OE-15 andOE-16 lines, which exhibited slight reductions. No apparent correlationwas found between the nicotine concentration and the NtERF98 or QPTexpression level.

D. Analysis of NtERF98 Transgenic RNAi Lines

A PCR fragment of 309 bp from NtERF98 coding sequence was used to makethe RNAi construct. Northern analysis was performed to evaluate whetherthe knockdown of NtERF98 has an effect on the QPT expression. FIG. 18shows that, out of 10 transformed lines, six had the NtERF98 expressioncompletely repressed (RNAi-6 through -12), and the other four hadslightly or moderately reduced expression.

Northern analysis of QPT gene expression of the NtERF98 RNAi lines wasperformed. FIG. 19 shows the QPT expression in the RNAi plants whencompared to a control plant. Various degree of reduction in QPTexpression was observed among the transgenic plants. However, whennicotine concentration was examined, seven out of ten RNAi plants hadmoderate increase in nicotine level (30-60% higher) with the other threehaving levels similar to that of the non-transgenic control (FIG. 20).

Overall, NtERF98 appears to play a role in modulating QPT expression. Itis intriguing that both over-expression and suppression of itsexpression generally led to a reduction of the QPT mRNA level and to aslight to moderate increase of nicotine concentration. The observationunderscores the complex nature of the regulation of the nicotinebiosynthesis pathway. Investigation of the relevant gene expression andnicotine level of the transgenic plants after topping or woundingtreatment may provide more insight into the role of NtERF98 in nicotinebiosynthesis.

Example 5. Transcription Factor NtETTa

The full length cDNA sequence of the transcription factor NtETTa genewas 2429 bp in length and encodes a protein of 739 AA. The cloned TFfactor gene was named NtETTa.

A. Expression Pattern of NtETTa in Tobacco Plant

The expression of the NtETTa gene in mature tobacco plant was examinedby Northern analysis. FIG. 21 shows that it has higher expression instem and low expression in root, leaf, and flower.

B. NtETTa Expression in Tobacco Root After Topping, Wounding, or MeJATreatment.

The three treatments were also applied to test the responses of theNtETTa gene expression in tobacco root. FIG. 22 shows a clearup-regulation of NtETTa gene expression 0.25 h after any of thesetreatments with mRNA level increased more by topping and wounding.However, unlike the MYC2 genes, no clear biphasic increase pattern wasobserved for NtETTa gene expression. MeJA seems to be the leasteffective treatment with respect to NtETTa induction.

C. Analysis of Transgenic Plants that Over- or Under-Express NtETTa Gene

Four NtETTa over-expression and three RNAi transgenic tobacco lines wereobtained. Northern blot analysis was undertaken to characterize theselines. FIG. 23 shows that all the four over-expression lines had higherNtETTa expression levels with OE-6 and OE-7 being much higher than thecontrols, and three RNAi lines showed almost no detectable expression.

To test the effect of NtETTa on QPT gene expression, Northern analysisof these over-expression and RNAi lines was performed. FIG. 24 showsthat both NtETTa over-expression lines and RNAi lines had lower QPTexpression levels when compared to the controls. Two RNAi lines (RNAi-1and RNAi-2) exhibited the lowest QPT expression level.

For all those lines, nicotine concentration in the leaf was determined.FIG. 25 shows that OE-4, -5 and RNAi-2, -3 had approximately 25% lowernicotine level than the controls while the nicotine concentration inOE-6 was slightly higher (25%).

The expression of the ARF transcription factor gene isolated in thisexperiment, NtETTa, was quickly induced by topping, wounding, and MeJAtreatment. It affects QPT expression as revealed in eitherover-expression or RNAi transgenic lines. It was shown in RNAi-1 and -2lines that suppression of NtETTa expression could lead to severereduction of QPT mRNA level but only limited change in nicotine level.This again points to the complicated control of nicotine biosynthesis,and to our previous observation that nicotine level may not becorrelated to QPT (and possible PMT) mRNA level. Additional experimentsto determine the effect of stimulations like topping, wounding, or MeJAtreatment on the transgenic plants (in terms of PMT and QPT mRNA levelsand the nicotine accumulation), should elucidate the role of NtETTa innicotine biosynthesis pathway further.

Example 6. Characterization of T3 Transgenic Plants

A. Greenhouse Grown T3 Plants (NC-GH-2013)

T3 plants of AOE and BOE events were grown in a greenhouse from seed ofT2 plants of events AOE3, AOE6, BOE16 and BOE17. The seeds were plantedin 288 cell trays in an improvised float system in greenhouses.Individual T3 plants of each subfamily were tested for the presence of aT-DNA insert gene by PCR analysis for the marker gene to determine thesegregation pattern. Non-segregating subfamilies were identified forevents AOE3 (AOE3-46-68), AOE6 (AOE6-30-77), and BOE16 (BOE16-37-93,BOE16-37-94) and plants from these subfamilies comprised 4 of the setsin the trial. Three additional sets included in the trial, AOE3-46(S),AOE6-30(S), and BOE17-12(S), consisted of PCR-positive plants fromsegregating seed lots. Two sets of control plants were included, a linetransformed with a vector containing no MYC2 coding sequence (VC), andplants from seed from a cross of the vector control with untransformedtobacco (VC×NT)

Each set consisted of three plants. Seedlings were transplanted to 4 galplastic bags with drainage holes punched in them containingapproximately 14 liters of potting mix/bag.

Pre-topping leaf samples were taken 13 weeks after transplanting, andthen the plants were topped. Additional leaf samples were taken 17 and31 days after topping. Leaf samples were allowed to dry at RT for 10days and then the leaf lamina were dried at 60° C. Dried leaf lamina wasanalyzed for total nicotinic alkaloids.

As shown in FIGS. 26A-26C, total alkaloid levels were higher in AOEplants both before and after topping. Total alkaloids in the BOE plantswere similar to control levels prior to topping, but increased aftertopping.

B. Field Grown T3 Plants (2013-VA-1)

T3 plants of AOE and BOE events described above, were grown in thefield. The trial included 10 sets of plants from the seedlings used inExample 6, part A, above, the two controls, VC and VC×NT, and 2 entriesfrom each of the four transgenic events AOE3, AOE6, BOE16 and BOE17.Four entries, AOE3-46-68, AOE6-30-77, BOE16-37-93, and BOE16-37-94, werecomposed of plants from non-segregating seed lots. Four entries,AOE3-46(S), AOE6-30(S), BOE17-12(S1), and BOE17-12(S2), were composed ofPCR-positive plants selected from segregating T3 seed lots. Each set wasplanted in 5 replicates of 3 plants each.

Leaf samples were taken prior to topping and 17 and 31 days aftertopping. Leaf samples were dried and total alkaloids in leaf laminameasured as in A.

As shown in the FIGS. 27A-27C, total alkaloid levels were higher in AOEplants both before and after topping.

Example 7. Discussion

QPT2 Promoter and Yeast One-Hybrid Screening

Among the major nicotine synthesis-related genes, only the promotersequence of NtPMT1a was analyzed in detail, and three basictranscription regulatory motifs were identified. They are a G-box(GCACGTTG, −103 to −96 bp from transcription initiation site), aGCC-like box (TGCGCCC, −62 to −56 bp) and an AT rich region in between(−80 to −69 bp, 92.8% A and T bases). These elements were demonstratedto be important in regulation of nicotine accumulation under JAtreatment, and were named GAG motif (Timko et al. (2010). RecentAdvances in Tobacco Science 36: 25-39). Although the GAG motif was foundin all NtPMT gene promoters, a BLAST search could not identify such amotif in other gene promoters in public available database (Id.),suggesting that the motif is a critical element required to coordinateexpression of the NtPMT family members. It also implies that theexpression of other nicotine synthesis pathway genes may not beregulated in the same way although their expression has to be somewhatcoordinated to produce nicotine efficiently. The tobacco QPT2 genepromoter was used in this study as a bait to isolate transcriptionfactors that bind to the QPT2 promoter and regulate its gene expression.The disadvantage is that the QPT2 gene promoter has not previously beenanalyzed and characterized in detail. A sequence scanning analysis ofthe promoter found that a “G-box” sequence (AACGTG) lies at −205 bpupstream of the translational start site ATG as predicted by theweb-based software (plantCARE,bioinformatics.psb.ugent.be/webtools/plantcare/html/).

In the yeast one-hybrid system, a library of proteins fused with theactivation domain of GAL4 are screened by their binding to the baitpromoter sequence and activates the reporter gene expression (His3 inthis case). As recommended in the user manual, at least three tandemcopies of the target sequence (bait) should be included in the baitvector upstream of the reporter gene to increase the chance for binding.Usually, cis-elements less than 20 bp are used to make the tandem copiesand the total bait is around 100 bp long. In this study, because the QPTpromoter region was not well characterized, the 1 kb QPT2 gene promoterwas used as a bait. A total of around 100 positive colonies wereisolated and their sequences were determined and analyzed. Three out offive TF genes isolated from the system have roles in regulating genes inthe nicotine biosynthesis pathway.

Isolation of Four Transcription Factors Involved in NicotineBiosynthesis

A total of four TFs were isolated, which are involved in the nicotinebiosynthesis pathway. They modulate the QPT mRNA level and affectnicotine concentration. Their expression in roots is responsive totopping, wounding, and MeJA treatments in a similar manner: theirexpression levels change as quickly as within 15 min upon thetreatments. NtMYC2a and 2b are positively induced by these treatmentsand showed two induction peaks within the first six hours aftertreatments. In contrast, whereas NtERF98 mRNA level was negativelyregulated and exhibited biphasic decreases within six hours, NtETTaexpression was also induced by the treatments within 15 min but did notshow a clear biphasic pattern. The long-distance movement signal thatcauses such rapid changes (within 15 min) in gene expression level inroots is still unknown. Neither JA nor auxin seems to move that fastwithin plants (Hertel and Flory (1968) Planta 82: 123-144; Baldwin etal. (1997) Planta 201: 397-404; Shi et al. (2006) J Exp Bot 57:2899-2907). From another perspective, wounding can induce production ofsmRNA. Recent research shows that silenced RdR1 (RNA directed RNApolymerase) in Nicotiana attenuate made the plants susceptible toherbivores (Pandey et al. (2008) Proc Natl Acad Sci USA 105:4559-4564).This result suggests that the smRNA is involved in the wounding-inducedresponse of tobacco plant and may be a fast-moving signal candidate.

The fact that these TF genes are expressed in all the tissues tested isnot surprising. Each of these TFs are likely involved in the JAsignaling pathway, which regulates not only nicotine (or otheralkaloids) biosynthesis, but also many other physiological anddevelopmental processes, such as root growth, fertility, resistance todiseases and even responses to abiotic stress like drought (Kazan andManners (2008) Plant Physiol 146: 1459-1468). Interestingly, despitetheir potential multiple roles in plant growth, transgenic plants withconstitutive over-expression or down-regulation of each of these four TFgenes all appeared normal when growing in greenhouse.

Transcription Factors NtMYC2a, NtMYC2b are Positive Regulators ofNicotine Biosynthesis

MYC2 has been considered the core TF in JA signaling pathway byregulating a cascade of transcription factors in plant responses to JA.It is known that MYC2 binds to the G-box motif of a promoter (Dombrechtet al. (2007) Plant Cell 19: 2225-2245). Because all the PMT genepromoters contain the G-box (Timko et al. (2010) Recent Advances inTobacco Science 36: 25-39) and the QPT promoter has a “G-box” sequence,it is likely that MYC2 directly regulates expression of these two keygenes. In two over-expression lines of NtMYC2a, the constitutivenicotine level (untreated) was more than doubled attaining about 1% ofleaf dry weight, which is very high. NtMYC2b also facilitated anapproximately 50% increase in nicotine level in three over-expressionlines. In contrast, three RNAi lines, in which it is likely that bothNtMYC2a and b were down-regulated (the probe used in Northern blotanalysis cannot distinguish the two), had approximately a five-folddecrease in nicotine level accompanied by approximate 10-fold decreasein mRNA levels of PMT and QPT genes. All indicate a positive regulatorrole of NtMYC2a and b in nicotine biosynthesis.

Notably, a dramatic decrease of both PMT and QPT mRNA levels in theNtMYC2a was observed in the over-expression lines. This may indicate anegative feedback loop when the nicotine level reaches a certainthreshold level to prevent an uncontrolled accumulation (Kazan andManners (2008) Plant Physiol 146: 1459-1468). Alternatively, althoughthe mRNAs of PMT and QPT were down-regulated by an unknown mechanismrelated to NtMYC2a over-expression, their protein levels were increasedor their enzymes were more active to enhance nicotine production. Inaddition, because PMT and QPT mRNA levels were not affected as much inthe NtMYC2b over-expression lines, these results also reveal a possiblediverse function between the 2a and 2b isoforms. The phenomenon may beexplained by a negative regulatory loop found in Arabidopsis. Here,AtMYC2 was demonstrated to up-regulate repressor JAZ protein, which inturn binds, and represses the activity of, AtMYC2 (Staswick (2007)Trends in Plant Sci 13: 66-71). Similarly, high levels of NtMYC2bwouldn't be able to activate the PMT and QPT expression if itup-regulates NtJAZ expression.

Overall, NtMYC2b seems to be the “weaker” isoform between the twobecause in its over-expression lines its mRNA levels increasedapproximately 10-fold and the nicotine level was only enhanced by about50%.

The TF genes, NbbHLH1 and NbbHLH2, from the same subgroup of the bHLHfamily were recently isolated and characterized in N. benthamiana (Toddet al. (2010) Plant J 62: 589-600). NbbHLH1 shares 70% AA homology withthe NtMYC2b while NbbHLH2 has 96% AA identity with NtMYC2a gene. NbbHLH1and 2 bind at the G-box sequence of the NbPMT gene promoter, andpositively regulate nicotine biosynthesis in transgenic tobacco plants.However, Todd et al. (Id.) were also puzzled by the observation oflittle change or even reduction of mRNA levels of most nicotinebiosynthesis genes, including QPT, in NbbHLH1 and NbbHLH2over-expression transgenic tobacco plants. This report verifies that ourobservation was not an exception or experimental error, and points to amore complicated regulatory network in nicotine biosynthesis pathway.

Zhang et al. (2012) also reported isolation and characterization ofthree related NtMYC2 genes: NtMYC2a, 2b and 2c, among which 2b and 2chave identical amino acid sequences but are diverse in nucleotidesequence. However, after AA sequence examination, the NtMYC2a and 2bthey reported (GenBank No. HM466974 and HM466975) have been shown to bethe same transcription factors as NtMYC1b (ADH04268) and NtMYC1a(ADH04267), respectively, and not NtMYC2a and NtMYC2b.

Possible Roles of Transcription Factors NtERF98 and NtETTa in NicotineBiosynthesis

The ethylene and JA signaling interaction is rather complex and could beboth synergistic and antagonistic (Kazan and Manners (2008) PlantPhysiol 146: 1459-1468). Both repressor- and activator-type ethylenereaction factors (ERFs) have been reported (McGrath et al. (2005) PlantPhysiol 139: 949-959). In tobacco, ethylene is shown to have negativeeffects on nicotine biosynthesis (Wang et al. 1994; Shoji et al. (2000)Plant Cell Physiol 41: 1072-1076; Winz and Baldwin (2001) Plant Physiol125: 2189-2202). Our research revealed that NtERF98 has a rathercomplicated role in nicotine biosynthesis. NtERF98 is a negativeregulator of nicotine synthesis in that its expression is down-regulatedby all three treatments that stimulate nicotine synthesis and that thenicotine levels increased by up to 50% in a majority of its RNAi lines.In addition, there was a good negative correlation between NtERF98 andQPT mRNA levels in most of the over-expression lines. However, in thoselines, while QPT was down-regulated, nicotine level was unchanged orincreased only moderately. Moreover, in the RNAi lines where nicotinelevels were generally increased, the QPT mRNA levels were reducedslightly. Overall NtERF98 may have a modifying function on nicotinelevel but may be more responsible for modulating QPT mRNA level. This TFmay be involved in the tight control of QPT mRNA level

The auxin and JA signaling pathways are closely interlinked. It has beenshown that one phytohormone activates biosynthesis genes of the otherand vice versa. Moreover, at least two ARFs are required for JAbiosynthesis and plant fertility (for review, see Kazan and Manners(2008) Plant Physiol 146: 1459-1468). Recently it was reported that theJAZ1 repressor gene is activated by both JA and auxin (Grunewald et al.(2009) EMBO Rep. 10: 923-928). In the present invention, the cloned ARFTF gene, NtETTa, appears to positively regulate QPT expression as shownin the RNAi lines where QPT mRNA was reduced approximately 15 fold,indicating that NtETTa is required for QPT expression. However,over-expression of NtETTa did not increase QPT expression and oftenmoderately reduced its mRNA level, implicating a role of NtETTa in thecomplicated tight control of QPT mRNA level.

Recently, an auxin responsive transcription factor, NbARF 1, wasreported as a negative regulator in nicotine synthesis. Suppression ofNbARF 1 by VIGS significantly enhanced nicotine level in untreatedplants (Todd et al. (2010) Plant J 62: 589-600). NtETTa is an auxinresponsive transcription factor but it acts as a positive regulator onQPT expression, although it appears to have minor effects on nicotinelevel.

In conclusion, four TF genes were cloned in this study. They are NtMYC2aand NtMYC2b from the bHLH family, NtERF98 from the AP2/ERF family, andNtETTa from the ARF family. They are all involved in regulation of rootQPT mRNA level and/or leaf nicotine level, with NtMYC2a and NtMYC2bhaving a more positive effects, and NtERF98 and NtETTa being rather morecomplicated modulators. NtMYC2a appears to play a more important role inregulating nicotine synthesis: Over-expression increases constitutivenicotine level by up to 2.5-fold whereas down-regulation of the gene(together with NtMYC2b) reduces it by five fold. Our research alsorevealed that higher nicotine synthesis is not always associated withhigher mRNA levels of a key pathway gene, QPT, pointing to a negativefeedback loop and/or possible translational and/or posttranslationalcontrol in the pathway.

The foregoing is illustrative of the invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

APPENDIXNucleotide and amino acid sequences of the transcription factors isolatedin this research NtMYC2a cDNA (full-length, 2214 bp)CACACACTCTCTCCATTTTCACTCACTCCTTATCACCAAACAATTCTTGGGTGTTTGAATATATACCCGAAATAATTTCCTCTCTGTATCAAGAATCAAACAGATCTGAATTGATTTGTCTGTTTTTTTTTCTTGATTTTGTTATATGGAATGACGGATTATAGAATACCAACGATGACTAATATATGGAGCAATACTACATCCGATGATAATATGATGGAAGCTTTTTTATCTTCTGATCCGTCGTCGTTTTGGCCCGGAACAACTACTACACCAACTCCCCGGAGTTCAGTTTCTCCAGCGCCGGCGCCGGTGACGGGGATTGCCGGAGACCCATTAAAGTCTATGCCATATTTCAACCAAGAGTCACTGCAACAGCGACTCCAGACTTTAATCGATGGGGCTCGCAAAGGGTGGACGTATGCCATATTTTGGCAATCGTCTGTTGTGGATTTCGCGAGCCCCTCGGTTTTGGGGTGGGGAGATGGGTATTATAAAGGTGAAGAAGATAAAAATAAGCGTAAAACGGCGTCGTTTTCGCCTGACTTTATCACGGAACAAGCACACCGGAAAAAGGTTCTCCGGGAGCTGAATTCTTTAATTTCCGGCACACAAACCGGTGGTGAAAATGATGCTGTAGATGAAGAAGTAACTGATACTGAATGGTTTTTTCTGATTTCCATGACACAATCGTTTGTTAACGGAAGCGGGCTTCCGGGCCTGGCGATGTATAGTTCAAGCCCGATTTGGGTTACTGGAACAGAGAGATTAGCTGTTTCTCACTGTGAACGGGCCCGACAGGCCCAAGGTTTCGGGCTTCAGACTATTGTTTGTATTCCTTCAGCTAATGGTGTTGTTGAGCTCGGGTCAACTGAGTTGATATTCCAGACTGCTGATTTAATGAACAAGGTTAAAGTTTTGTTTAATTTTAATATTGATATGGGTGCGACTACGGGCTCAGGATCGGGCTCATGTGCTATTCAGGCCGAGCCCGATCCTTCAGCCCTTTGGCTGACTGATCCGGCTTCTTCAGTTGTGGAAGTCAAGGATTCGTCGAATACAGTTCCTTCAAGGAATACCAGTAAGCAACTTGTGTTTGGAAATGAGAATTCTGAAAATGGTAATCAAAATTCTCAGCAAACACAAGGATTTTTCACTAGGGAGTTGAATTTTTCCGAATATGGATTTGATGGAAGTAATACTCGGTATGGAAATGGGAATGCGAATTCTTCGCGTTCTTGCAAGCCTGAGTCTGGTGAAATCTTGAATTTTGGTGATAGTACTAAGAGGAGTGCTTGCAGTGCAAATGGGAGCTTGTTTTCGGGCCAATCACAGTTCGGGCCCGGGCCTGCGGAGGAGAACAAGAACAAGAACAAGAAAAGGTCACCTGCATCAAGAGGAAGCAACGATGAAGGAATCCTTTCATTTGTTTCGGGTGTGATTTTGCCAAGTTCAAACACGGGGAAGTCCGGTGGAGGTGGCGATTCGGATCAATCAGATCTCGAGGCTTCGGTGGTGAAGGAGGCGGATAGTAGTAGAGTTGTAGACCCCGAGAAGAAGCCGAGGAAACGAGGGAGGAAACCGGCTAACGGGAGAGAGGAGCCATTGAATCATGTGGAGGCAGAGAGACAAAGGAGGGAGAAATTGAATCAAAGATTCTATGCACTTAGAGCTGTTGTACCAAATGTGTCAAAAATGGATAAAGCATCACTTCTTGGTGATGCAATTGCATTTATCAATGAGTTGAAATCAAAGGTTCAGAATTCTGACTCAGATAAAGAGGACTTGAGGAACCAAATCGAATCTTTAAGGAATGAATTAGCCAACAAGGGATCAAACTATACCGGTCCTCCCCCGTCAAATCAAGAACTCAAGATTGTAGATATGGACATCGACGTTAAGGTGATCGGATGGGATGCTATGATTCGTATACAATCTAATAAAAAGAACCATCCAGCCGCGAGGTTAATGACCGCTCTCATGGAATTGGACTTAGATGTGCACCATGCTAGTGTTTCAGTTGTCAACGAGTTGATGATCCAACAAGCGACTGTGAAAATGGGAAGCCGGCTTTACACGCAAGAACAACTTCGGATATCATTGACATCCAGAATTGCTGAATCGCGATGAAGAGAAATACAGTAAATGGAAATTATCATAGTGAGCTCTGAATAATGTTATCTTTCATTGAGCTATTTTAAGAGAATTTCTCCT AAAAAAAAAAAAAAAAAAAAAAAAAAA SEQ ID NO: 1NtMYC2a amino acid sequence (659 AA)M T D Y R I P T M T N I W S N T T S D D N M M E A F L S S D P S S F W P G T T T TP T P R S S V S P A P A P V T G I A G D P L K S M P Y F N Q E S L Q Q R L Q T L I DG A R K G W T Y A I F W Q S S V V D F A S P S V L G W G D G Y Y K G E E D K N KR K T A S F S P D F I T E Q A H R K K V L R E L N S L I S G T Q T G G E N D A V D EE V T D T E W F F L I S M T Q S F V N G S G L P G L A M Y S S S P I W V T G T E R LA V S H C E R A R Q A Q G F G L Q T I V C I P S A N G V V E L G S T E L I F Q T A DL M N K V K V L F N F N I D M G A T T G S G S G S C A I Q A E P D P S A L W L T DP A S S V V E V K D S S N T V P S R N T S K Q L V F G N E N S E N G N Q N S Q Q TQ G F F T R E L N F S E Y G F D G S N T R Y G N G N A N S S R S C K P E S G E I L NF G D S T K R S A C S A N G S L F S G Q S Q F G P G P A E E N K N K N K K R S P AS R G S N D E G I L S F V S G V I L P S S N T G K S G G G G D S D Q S D L E A S V VK E A D S S R V V D P E K K P R K R G R K P A N G R E E P L N H V E A E R Q R R EK L N Q R F Y A L R A V V P N V S K M D K A S L L G D A I A F I N E L K S K V Q NS D S D K E D L R N Q I E S L R N E L A N K G S N Y T G P P P S N Q E L K I V D M DI D V K V I G W D A M I R I Q S N K K N H P A A R L M T A L M E L D L D V H H A SV S V V N E L M I Q Q A T V K M G S R L Y T Q E Q L R I S L T S R I A E S RSEQ ID NO: 2 NtMYC2b cDNA (full-length, 2391 bp)GTAACAAACCCTCTCCATTTTCACTCACTCCAAAAAACTTTCCTCTCTATTTTTTCTCTCTGTATCAAGAATCAAACAGATCTGAATTGATTTGGGAGTTTTTTTTCTTCTTGTTTTTGTTATATGGAATGACGGACTATAGAATACCAACGATGACTAATATATGGAGCAATACAACATCCGACGATAACATGATGGAAGCTTTTTTATCTTCTGATCCGTCGTCGTTTTGGGCCGGAACAAATACACCAACTCCACGGAGTTCAGTTTCTCCGGCGCCGGCGCCGGTGACGGGGATTGCCGGAGACCCATTAAAGTCGATGCCGTATTTCAACCAAGAGTCGCTGCAACAGCGACTCCAGACGTTAATCGACGGGGCTCGCGAAGCGTGGACTTACGCCATATTCTGGCAATCGTCTGTTGTGGATTTCGTGAGCCCCTCGGTGTTGGGGTGGGGAGATGGATATTATAAAGGAGAAGAAGACAAGAATAAGCGTAAAACGGCGGCGTTTTCGCCTGATTTTATTACGGAGCAAGAACACCGGAAAAAAGTTCTCCGGGAGCTGAATTCTTTAATTTCCGGCACACAAACTGGTGGTGAAAATGATGCTGTAGATGAAGAAGTAACGGATACTGAATGGTTTTTTCTGATTTCAATGACTCAATCGTTTGTTAACGGAAGCGGGCTTCCGGGCCTGGCTATGTACAGCTCAAGCCCGATTTGGGTTACTGGAAGAGAAAGATTAGCTGCTTCTCACTGTGAACGGGCCCGACAGGCCCAAGGTTTCGGGCTTCAGACTATGGTTTGTATTCCTTCAGCTAATGGTGTTGTTGAGCTCGGGTCAACTGAGTTGATATTCCAGAGCGCTGATTTAATGAACAAGGTTAAAATCTTGTTTGATTTTAATATTGATATGGGCGCGACTACGGGCTCAGGTTCGGGCTCATGTGCTATTCAGGCTGAGCCCGATCCTTCAACCCTTTGGCTTACGGATCCACCTTCCTCAGTTGTGGAAGTCAAGGATTCGTCGAATACAGTTCCTTCAAGTAATAGTAGTAAGCAACTTGTGTTTGGAAATGAGAATTCTGAAAATGTTAATCAAAATTCTCAGCAAACACAAGGATTTTTCACTAGGGAGTTGAATTTTTCCGAATATGGATTTGATGGAAGTAATACTAGGAGTGGAAATGGGAATGTGAATTCTTCGCGTTCTTGCAAGCCTGAGTCTGGCGAAATCTTGAATTTTGGTGATAGTACTAAGAGAAATGCTTCAAGTGCAAATGGGAGCTTGTTTTCGGGCCAATCGCAGTTCGGTCCCGGGCCTGCGGAGGAGAACAAGAACAAGAACAAGAAAAGGTCACCTGCATCAAGAGGAAGCAATGAAGAAGGAATGCTTTCATTTGTTTCGGGTGTGATCTTGCCAAGTTCAAACACGGGGAAGTCCGGTGGAGGTGGCGATTCGGATCATTCAGATCTCGAGGCTTCGGTGGTGAAGGAGGCGGATAGTAGTAGAGTTGTAGACCCCGAGAAGAGGCCGAGGAAACGAGGAAGGAAACCGGCTAACGGGAGAGAGGAGCCATTGAATCATGTGGAGGCAGAGAGGCAAAGGAGGGAGAAATTGAATCAAAGATTCTATGCACTTAGAGCTGTTGTACCAAATGTGTCAAAAATGGATAAAGCATCACTTCTTGGTGATGCAATTGCATTTATCAATGAGTTGAAATCAAAGGTTCAGAATTCTGACTCAGATAAAGATGAGTTGAGGAACCAAATTGAATCTTTAAGGAATGAATTAGCCAACAAGGGATCAAACTATACCGGTCCTCCACCGCCAAATCAAGATCTCAAGATTGTAGATATGGATATCGACGTTAAAGTCATCGGATGGGATGCTATGATTCGTATACAATCTAATAAAAAGAACCATCCAGCCGCGAGGTTAATGGCCGCTCTCATGGAATTGGACTTAGATGTGCACCATGCTAGTGTTTCAGTTGTCAACGAGTTGATGATCCAACAAGCGACAGTGAAAATGGGGAGCCGGCTTTACACGCAAGAGCAGCTTCGGATATCATTGACATCCAGAATTGCTGAATCGCGATGAAGAGAAATACAGTAAATGGAAATTATTAGTGAGCTCTGAATAATGTTATCTTTCATTGAGCTATTTTAAGAGAATTTCTCCTATAGTTAGATCTTGAGATTAAGGCTACTTAAAAGTGGAAAGTTGATTGAGCTTTCCTCTTAGTTTTTTGGGTATTTTTCAACTTTTATATCTAGTTTGTTTTCCACATTTTCTGTACATATAATGTGAAACCAATACTAGATCTCAAGATCTGGTTTTTAGTTCTGTAATTAGAAATAAATATGCAGCTTCATCTTTTTCTGTTAAAAAAAAAAAAAAAAAAAAAAAAA SEQ ID NO: 3 NtMYC2b amino acid sequence (658 AA)M T D Y R I P T M T N I W S N T T S D D N M M E A F L S S D P S S F W A G T N T PT P R S S V S P A P A P V T G I A G D P L K S M P Y F N Q E S L Q Q R L Q T L I D GA R E A W T Y A I F W Q S S V V D F V S P S V L G W G D G Y Y K G E E D K N K RK T A A F S P D F I T E Q E H R K K V L R E L N S L I S G T Q T G G E N D A V D E EV T D T E W F F L I S M T Q S F V N G S G L P G L A M Y S S S P I W V T G R E R L AA S H C E R A R Q A Q G F G L Q T M V C I P S A N G V V E L G S T E L I F Q S A D LM N K V K I L F D F N I D M G A T T G S G S G S C A I Q A E P D P S T L W L T D P PS S V V E V K D S S N T V P S S N S S K Q L V F G N E N S E N V N Q N S Q Q T Q GF F T R E L N F S E Y G F D G S N T R S G N G N V N S S R S C K P E S G E I L N F GD S T K R N A S S A N G S L F S G Q S Q F G P G P A E E N K N K N K K R S P A S RG S N E E G M L S F V S G V I L P S S N T G K S G G G G D S D H S D L E A S V V KE A D S S R V V D P E K R P R K R G R K P A N G R E E P L N H V E A E R Q R R E K XL N Q R F Y A L R A V V P N V S K M D K A S L L G D A I A F I N E L K S K V Q N SD S D K D E L R N Q I E S L R N E L A N K G S N Y T G P P P P N Q D L K I V D M D ID V K V I G W D A M I R I Q S N K K N H P A A R L M A A L M E L D L D V H H A SV S V V N E L M I Q Q A T V K M G S R L Y T Q E Q L R I S L T S R I A E S RSEQ ID NO: 4 NtERF98 cDNA (full-length, 1019 bp)CACCGTCTCTTTCCATTTCTTTCTCTTAAAAGAAAAAACATCTCAATAACAAAAAGAAAAATGTGTGGAGGTGCCATAATCCCCGACTATGAACCCGTCGGAAACCGCTGCCGGAAAATCACTGCTAGTGACCTCTGGGCTGAGCTTGACCCTATCTCCGACTTCTGGTCTTCCTCTTCCTCTTCCTCCTCCATTGCCGGCAAATCTGATTCCGTTCAGTCGCTAACCCACTCCTACAATAAGCCTCAGAAATCAGATTCCGGCAAACTTAATCAACTCGAAAAAGGTACAATAAGTGTGAAGGTTGAGAAGGAGAGCAGTGGCCCAAGGGCGAGGAAGAACAAATACAGAGGAATAAGGCAGAGACCGTGGGGAAAATGGGCTGCTGAGATACGTGATCCTCAGAAAGGCGTCCGCGTGTGGTTAGGTACATTCAACACGGCTGAGGAAGCTGCCAGGGCATATGACGAGGCTGCAAAGCGAATCCGCGGTGACAAGGCTAAGCTCAACTTTCCAGAGCCACCTTCGCCACCAGCCAAGCGACACTGCACTAGCACCATCCCTGATCAGCCCACACGTTCTGACTTAATGTCTCAGAAACCGGCCTCAATAATGTTGAACTATGGATATGAAAACCAAACACCCTACTACCCCATGGAAATGCCCGCTGCTGAGGATCCTCAACATCATGATTATGAGCTCAAGGAGCAGATTTCCAACTTGGAGTCATTCCTGGATTTAGAGCCAGACTCAGGGATCGTCGATTCTGACCCCCTCAATATTTTTCTGATGGATGACTTTGCTGCAACTCAGCAGCAGCAGCTGTTTTACTGAACACTGTAAAAATTATCATATACTACTAGTTAATTTCATCCTAAGTTGTTTGGTGTGCGTTTTCTGATGAGTGACTAGTTAGCTTTTGGTAGTACGTAGTACAATGCAGAAAGTACATACAATAATAAGTTGCGTGCCTTTGCATGCAATTTGTAATATTAATGTCATGTTGTTTTGTGCTGTTTAAAAAAAAAAAAAAAAAAA SEQ ID NO: 5NtERF98 amino acid sequence (257 AA)M C G G A I I P D Y E P V G N R C R K I T A S D L W A E L D P I S D F W S S S S S SS S I A G K S D S V Q S L T H S Y N K P Q K S D S G K L N Q L E K G T I S V K V E KE S S G P R A R K N K Y R G I R Q R P W G K W A A E I R D P Q K G V R V W L G TF N T A E E A A R A Y D E A A K R I R G D K A K L N F P E P P S P P A K R H C T S TI P D Q P T R S D L M S Q K P A S I M L N Y G Y E N Q T P Y Y P M E M P A A E D PQ H H D Y E L K E Q I S N L E S F L D L E P D S G I V D S D P L N I F L M D D F A AT Q Q Q Q L F Y SEQ ID NO: 6 NtETTa cDNA (full-length, 2429 bp)AGCAAAAGGGTTTGAAGATGATGTGTGGACTTATTGATCTAAATACTGTGGATAACGATGACGTCGGAGAAGAAACGACGGCGCCGGTGTCACCAGCGTCATCGTCGACGGCGTCTGGATGTTCGGATTTGACGTCGTCATCTCTGCCGGCGATGGCATCGGTTTGTCTGGAGCTGTGGCATGCGTGTGCTGGACCGTTGATTTCTCTGCCGAAGAAAGGAAGTGCTGTTGTGTACCTACCTCAAGGTCACTTGGAACATCTCTCTGAGTACCCGCCCATAGCCTATAACCTCCCTCCTCACGTTTTTTGTCGCGTCGTAGACGTGAAGCTACAAGCGGATGCGGCGAGTGATGAGGTCTATGCACAAGTCTCACTGGTTCCAGACAATCAGATTGAGCAGAAATGGAGGGATGGAGACATTGATGCAGATACTGAAGAGGAGGAAATAGAAGGTGCTGGAAAATCAACAACACCACACATGTTCTGCAAGACTCTCACTGCTTCGGATACCAGCACTCATGGCGGTTTTTCTGTCCCTCGCCGGGCTGCAGAAGATTGCTTTCCTCCATTGGATTACAGACAACAGCGGCCCTCACAGGAGCTGGTAGCCAAAGATCTACATGGTATCGAGTGGAAATTTCGGCATATCTATCGTGGTCAGCCACGAAGGCATCTGCTCACTACAGGATGGAGTGCGTTTGTAAACAGGAAGAAGCTTGTTTCTGGTGACGCTGTGCTTTTCTTAAGGACTGCTGATGGAGAACTTAGGCTAGGGGTGAGACGAGCTGCCCAAGCTAAAACATGTTCAAATTATCTAGCTGCCTATAGCCAACTGTTGAATGTCAGTGGTATTGTGGATGTGGTTAAGGCCATATCTAGCACAAATGCCTTCAGTATCTGTTATAACCCGAGGGCTAGCTCATCAGGCTTCATTTTACCTTACCACAAATTCTCAAAGACTCTTGCACATCCCTTTTCAGCTGGAATGAGATTTAAGATGCGTGTCGAAACAGAAGATGCAGCTGAACAAAGGTTCACTGGACTTGTTGTAGGAGTCAGCGATGTAGATCCAGTTCGCTGGCCTGGTTCTAAATGGAGGTGCCTATTGGTCAGGTGGGATGATCTTGATGTTTCTCGGCATAATAGGGTTTCACCGTGGGAAATTGAGCCATCTGGTTCAGCTCCTGTATCCAGCAGCTTGGTGATGCCTTCTGCGAAGAGGACCAGGGTTGGCTTTCCAATTACAAAGGCCGATTTTCCAATTCCTAGAGATGGGATTGCAGTATCAGACTTTGGGGAATCTTCTAGGTTCCAGAAGGTCTTGCAAGGTCAAGAAATTTTGGGGATTAGTTCTCCTTTTGTCGGTTTTGATGCTCACAGTCCTCGTACAGCGGGGATAAGATGCTTTCCTGGTTTTCCTAGTTCTGGGGCATCTAGATTGGGAAACAGCATCAGAACCCTGCTTGGTGACACAGACAAGTCCCCTGAAAGCATTGGCTTTAGTGATTCTTCTCGATACAATAAGGTCTTGCAAGGTCAAGAAACTTTTTCAACCCCTCCTTATGGGAGAGGTCATGCAGGTAGCCTAATGCAGGAAAAAAGTAGAACTGGTATTATCGTCGGTATTCAGGTTCCAAGCCACGTAAACAGGTGGTCTGCTCCAAATCAGGGTAATCGCAGTCATTGCAATCCAAGTACTCTTGTCCCGGCATCATCACCTCCTTCTGTGCTCAGCTTTCAGCCTCCCAGGTCTCCAGCATCAAAATTCCAGGCTATGTTCAATCATAAACATGGGAAGCTTGAGACTGCTACCCAGGCTTTGGATATGTCTGAGAGCTGTAGTAGGCATCTCGCATCTGGCTCACATGCCGAGGACATCAGTAGGAAGGGAGACACAAAAGGAATCAGTTCTTTTAGTTTCTTAAAGGAGCAAAAGCAAACAGGAATTTCATATCTTTCTCTTGGGACCCAGTCGTCTCAAAACTTAGTTTCCATGTGTAAAACCAGTTGCAGGATCTTTGGATTCCCCTTGACCGAGAGTAAAATAAATGCAGCTAGAGCGGAGAATCCTGCCGAGGCTGTATATTCACATGGTCTAGAAACAACATTTCTGCCTTCCAGTGATGGAAAGTTGCAGCCAGGGCCACCATTGATGACTAATGTTGTGGGAACAAACTTTACTAAAGTAAATGACCTCTATGCTGCAAGAGATGTGATTCTTGATATTGCTTTGTAGCAAGTATTTGTTGTGAAGTCATGAGCATATGTAAACTGAAGGATGTGTGAGCAGTATTATTGATTCTTAGATTTTAGTTGGCTGATTAGTTTTGGCCAATGAACGCAAGCATGTAGTTGCCAGTACAATGCTTATCCTGAGATGAGTATTGAGAGTTTTTATTGTAAGGAACACAGTGAAGATTAGTATTGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAA SEQ ID NO: 7NtETTa amino acid sequence (739 AA)M M C G L I D L N T V D N D D V G E E T T A P V S P A S S S T A S G C S D L T S S SL P A M A S V C L E L W H A C A G P L I S L P K K G S A V V Y L P Q G H L E H L SE Y P P I A Y N L P P H V F C R V V D V K L Q A D A A S D E V Y A Q V S L V P D NQ I E Q K W R D G D I D A D T E E E E I E G A G K S T T P H M F C K T L T A S D T ST H G G F S V P R R A A E D C F P P L D Y R Q Q R P S Q E L V A K D L H G I E W KF R H I Y R G Q P R R H L L T T G W S A F V N R K K L V S G D A V L F L R T A D GE L R L G V R R A A Q A K T C S N Y L A A Y S Q L L N V S G I V D V V K A I S S TN A F S I C Y N P R A S S S G F I L P Y H K F S K T L A H P F S A G M R F K M R V ET E D A A E Q R F T G L V V G V S D V D P V R W P G S K W R C L L V R W D D L DV S R H N R V S P W E I E P S G S A P V S S S L V M P S A K R T R V G F P I T K A DF P I P R D G I A V S D F G E S S R F Q K V L Q G Q E I L G I S S P F V G F D A H S PR T A G I R C F P G F P S S G A S R L G N S I R T L L G D T D K S P E S I G F S D S SR Y N K V L Q G Q E T F S T P P Y G R G H A G S L M Q E K S R T G I I V G I Q V P SH V N R W S A P N Q G N R S H C N P S T L V P A S S P P S V L S F Q P P R S P A S KF Q A M F N H K H G K L E T A T Q A L D M S E S C S R H L A S G S H A E D I S R KG D T K G I S S F S F L K E Q K Q T G I S Y L S L G T Q S S Q N L V S M C K T S C R IF G F P L T E S K I N A A R A E N P A E A V Y S H G L E T T F L P S S D G K L Q P GP P L M T N V V G T N F T K V N D L Y A A R D V I L D I A L SEQ ID NO: 8

That which is claimed is:
 1. An isolated nucleic acid moleculecomprising a cDNA sequence selected from the group consisting of: (a)the nucleotide sequence of SEQ ID NO: 7; (b) a nucleotide sequence thatis at least 95% identical to the nucleotide sequence of SEQ ID NO: 7;and (c) a nucleotide sequence that encodes a polypeptide having theamino acid sequence of SEQ ID NO:
 8. 2. A nucleic acid constructcomprising, in the 5′ to 3′ direction, a heterologous promoter operablein a plant cell and, positioned downstream from the promoter andoperatively associated therewith, the nucleic acid molecule of claim 1.3. The nucleic acid construct of claim 2, wherein the promoter is aconstitutive promoter, a tissue preferred promoter, or a tissue specificpromoter.
 4. A vector comprising the nucleic acid molecule of claim 1.5. A transgenic Nicotiana tabacum plant cell or bacterial cellcomprising the vector of claim
 4. 6. A transgenic Nicotiana tabacumplant comprising the plant cell of claim
 5. 7. A seed of the transgenicplant of claim 6 or a progeny plant produced from the seed, wherein theseed and progeny plant comprise in their genomes the vector.
 8. A cropcomprising a plurality of transgenic Nicotiana tabacum plants accordingto claim 6 or a transgenic progeny thereof, wherein the plurality oftransgenic plants is planted together in an agricultural field, and thetransgenic N. tabacum plants and transgenic progeny thereof comprise intheir genomes the vector.
 9. A tobacco product produced from thetransgenic Nicotiana tabacum plant of claim 6, wherein the tobaccoproduct comprises the vector.
 10. The tobacco product of claim 9,wherein the tobacco product is selected from the group consisting of asmoking cessation product, a cigarette, cigarette tobacco, cigartobacco, a cigar, pipe tobacco, chewing tobacco, snuff, snus, lozenges,leaf tobacco, shredded tobacco, cut tobacco, and any combinationthereof.
 11. A method of producing a transgenic Nicotiana tabacum planthaving decreased alkaloid content, comprising: (a) introducing into aNicotiana tabacum plant cell a nucleic acid construct comprising atleast 21 consecutive nucleotides of a cDNA molecule comprising anucleotide sequence selected from the group consisting of: (i) thenucleotide sequence of SEQ ID NO: 7; (ii) a nucleotide sequence that isat least 95% identical to the nucleotide sequence of SEQ ID NO: 7; and(iii) a nucleotide sequence that encodes a polypeptide having the aminoacid sequence of SEQ ID NO: 8; wherein the consecutive nucleotides arein sense orientation, antisense orientation, or both; and (b)regenerating the transgenic Nicotiana tabacum plant cell to produce atransgenic Nicotiana tabacum plant comprising the nucleic acidconstruct.
 12. The method of claim 11, further comprising suppressingwithin the plant cell at least one of NBB1, A622, putrescineN-methyltransferase (PMT), quinolinic acid phosphoribosyltransferase(QPT), ornithine decarboxylase (ODC), arginine decarboxylase (ADC), andN-methylputrescine oxidase (MPO).
 13. A transgenic Nicotiana tabacumplant produced by the method of claim 11, wherein the transgenic N.tabacum plant comprises a decreased alkaloid content as compared to anon-transgenic control plant grown under similar conditions.
 14. A cropcomprising a plurality of the transgenic Nicotiana tabacum plants ofclaim 13 or a transgenic progeny plant thereof, wherein the plurality oftransgenic plants is planted together in an agricultural field.
 15. Atobacco product comprising the transgenic Nicotiana tabacum plant ofclaim 13, and having a decreased level of alkaloids as compared to acontrol product comprising the non-transgenic control plant.
 16. Thetobacco product of claim 15, wherein the tobacco product is selectedfrom the group consisting of a smoking cessation product, a cigarette,cigarette tobacco, cigar tobacco, a cigar, pipe tobacco, chewingtobacco, snuff, snus, lozenges, leaf tobacco, shredded tobacco, cuttobacco, and any combination thereof.
 17. A tobacco plant regeneratedfrom a tobacco plant cell comprising a mutant allele of a gene encodinga transcription factor that modulates nicotine biosynthesis, wherein thetobacco plant cell is produced by a method comprising: (a) introducinginto a population of tobacco plant cells a reagent for site-directedmutagenesis comprising an oligonucleotide having a targeting sequencewith at least 95% sequence identity to SEQ ID NO: 7, or a complementthereof, thereby producing the tobacco plant cell comprising the mutantallele of the gene encoding the transcription factor that modulatesnicotine biosynthesis; and (b) regenerating a tobacco plant from thetobacco plant cell of step (a), thereby resulting in a tobacco plantcomprising tobacco plant cells comprising the mutant allele of the geneencoding the transcription factor that modulates nicotine biosynthesis.18. Seeds from or a progeny plant of the tobacco plant of claim 17,wherein the seeds or progeny plant comprise in their genomes the mutantallele.
 19. A crop comprising a plurality of the tobacco plants of claim17 or a progeny thereof, wherein the plurality of tobacco plants orprogeny thereof comprise in their genomes the mutant allele.
 20. Atobacco product produced from the plant of claim 17, wherein the productcomprises the mutant allele, and wherein the tobacco product is selectedfrom the group consisting of a smoking cessation product, a cigarette,cigarette tobacco, cigar tobacco, a cigar, pipe tobacco, chewingtobacco, snuff, snus, lozenges, leaf tobacco, shredded tobacco, cuttobacco, and any combination thereof.